Mirna modulators of thermogenesis

ABSTRACT

Provided are novel methods and compositions for the modulation of thermogenesis. Such methods are particularly advantageous in that they allow for the reduction of body fat in a subject without the subject having to adjust their caloric intake through dieting, modify their physical activity or undergo bariatric surgery. Accordingly, the methods of the invention are particularly useful for treating or preventing obesity. Also provided are methods of screening for novel agents that modulate the activity of thermogenic regulators.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 14/714,470, filed May 18, 2015, which is a continuation of U.S. patent application Ser. No. 13/826,775, filed Mar. 14, 2013, which claims priority to U.S. Provisional Patent Application No. 61/636,059, filed Apr. 20, 2012, and U.S. Provisional Patent Application No. 61/681,750, filed Aug. 10, 2012. The entire contents of each of the above-referenced disclosures are specifically incorporated herein by reference without disclaimer.

BACKGROUND OF THE INVENTION

Obesity has reached pandemic proportions, affecting all ages and socioeconomic groups. The World Health Organization estimated that in 2008, 1.5 billion adults aged 20 years and older were overweight and over 200 million men and 300 million women were obese. These figures are estimated to increase to 2.16 billion overweight and 1.12 billion obese individuals by 2030. Obesity is the source of lost earnings, restricted activity days, absenteeism, lower productivity at work (presenteeism), reduced quality of life, permanent disability, significant morbidity and mortality, and shortened lifespan. Indeed, the total annual economic cost of overweight and obesity in the United States and Canada caused by medical costs, excess mortality and disability was estimated to be about $300 billion in 2009. International studies on the economic costs of obesity have shown that they account for between 2% and 10% of total health care costs.

Obesity is the result of a chronic imbalance between energy intake and expenditure. This leads to storage of excess energy into adipocytes, which typically exhibit both hypertrophy (increase in cell size) and hyperplasia (increase in cell number or adipogenesis). The recent worsening of obesity is due to the combination of excessive consumption of energy-dense foods high in saturated fats and sugars, and reduced physical activity.

The current symptomatic medical treatments of obesity fail to achieve their long-term therapeutic goals, largely due to limited drug efficacy and patients' poor adherence with lifestyle changes and therapies. Several obesity drugs have been removed from the market for safety reasons and small molecules currently in development are struggling to gain regulatory approval because of their modest short-term efficacy and unknown safety profile. Presently, only restrictive and malabsorptive bariatric surgery can achieve significant long-term reduction of weight excess with some favorable cardiovascular benefits.

Accordingly, there is a need in the art for novel treatments for obesity.

SUMMARY OF THE INVENTION

Obesity is the consequence of a chronic imbalance of energy intake over expenditure, leading to the storage of excess energy inside white adipocytes. This disclosure features a novel treatment for obesity targeting peripheral adipocytes, including energy-storing lipid-filled white adipocytes (WAT), and energy-expending mitochondria-rich brown adipocytes (BAT). In addition, the disclosure provides methods for the modulation of thermogenesis (the process of heat production in organisms) using microRNA (miRNAs) agents. The methods described herein generally involve the direct and/or indirect modulation of at least one thermogenic regulator (e.g., a mitochondrial uncoupler, such as Uncoupling Protein 1 (UCP1 also known as Thermogenin) or Uncoupling Protein 2 (UCP2)) in a cell, tissue and/or subject using an isolated miRNA agent. UCPs uncouple oxidative phosphorylation from ATP synthesis. In certain instances, this uncoupling results in energy dissipated as heat. Such methods are particularly advantageous in that they allow for the reduction of body fat in a subject without the subject having to adjust their caloric intake through dieting, modify their physical activity or undergo bariatric surgery. Accordingly, the methods of the invention are particularly useful for treating or preventing obesity.

The invention also provides novel miRNA agent compositions (e.g., miRNA, agomirs, and antagomirs) that can modulate the activity of thermogenic regulators. Yet further, the invention provides methods of screening for novel miRNA agents that modulate the activity of thermogenic regulators. Further still, the invention provides novel agent compositions (e.g. aptamer-miRNA complexes or “aptamirs”) that provide cell/tissue-specific delivery of the miRNA agents.

Accordingly, in one aspect, the invention provides a method of modulating respiratory chain uncoupling in a cell, the method comprising contacting the cell with an isolated miRNA agent that modulates the expression level and/or activity of at least one mitochondrial uncoupler. In some embodiments, the method further comprises the step of selecting a subject in need of modulating respiratory chain uncoupling (e.g., an obese patient). In one embodiment, the miRNA agent increases the expression level and/or activity of the at least one mitochondrial uncoupler. In certain embodiments, the mitochondrial uncoupler is UCP1 or UCP2. In some embodiments, the method increases respiratory chain uncoupling in a cell in vivo. In other embodiments, the method increases respiratory chain uncoupling in a cell ex vivo. In certain embodiments, the method further comprises determining the level of expression (mRNA or protein) or activity of the mitochondrial uncoupler. In certain embodiments, the cell is a pre-adipocyte, adipocyte, adipose tissue derived mesenchymal stem cell, hepatocyte, myocyte, or a precursor thereof. Optionally, adipocytes can be white fat or brown fat adipocytes.

In another aspect, the invention provides a method of modulating thermogenesis in a tissue, the method comprising contacting the tissue with an isolated miRNA agent that modulates the expression level and/or activity of at least one mitochondrial uncoupler. In some embodiments, the method further comprises the step of selecting a subject in need of modulating thermogenesis (e.g., an obese patient). In one embodiment, the miRNA agent increases the expression level and/or activity of the at least one mitochondrial uncoupler. In certain embodiments, the mitochondrial uncoupler is UCP1 or UCP2. In certain embodiments, the method involves increasing thermogenesis. In certain embodiments, the method further comprises determining the level of expression (mRNA or protein) or activity of the mitochondrial uncoupler. In certain embodiments, the tissue is brown fat, white fat, subcutaneous adipose tissue, liver or muscle. In certain embodiments, the tissue is contacted with the miRNA agent ex vivo.

In another aspect, the invention provides a method of treating obesity in human subject in need of treatment thereof, the method generally comprising administering to the human subject an effective amount of a miRNA agent that modulates activity of at least one mitochondrial uncoupler. In certain embodiments, the human subject selected for treatment has a genetic or epigenetic predisposition to obesity. In certain embodiments, the mitochondrial uncoupler is UCP1 or UCP2.

In certain embodiments of all of the above aspects, the miRNA agent is an isolated miRNA selected from the group consisting of hsa-miR-1-1, hsa-miR-1-2, miR-19a-b, hsa-miR-105, hsa-miR-1283, hsa-mir-129, hsa-miR-133a-1, hsa-miR-133a-2, hsa-miR-143, hsa-mir-143-5p, hsa-mir-147, hsa-mir-149, hsa-mir-199a, hsa-mir-199b, hsa-mir-200c, hsa-mir-204, hsa-mir-205, hsa-miR-206, hsa-mir-21, hsa-mir-211, hsa-mir-218, hsa-mir-218-1, hsa-mir-218-2, hsa-mir-219-2, hsa-mir-219-2-3p, hsa-mir-22, hsa-mir-22-3p, hsa-mir-22-5p, hsa-mir-24-2, hsa-miR-30a-e, hsa-miR-3177-5p, hsa-mir-325, hsa-mir-331, hsa-mir-331-5p, hsa-miR-3613-3p, hsa-mir-362, hsa-mir-362-5p, hsa-miR-3658, hsa-mir-367, hsa-mir-371, hsa-mir-371-5p, hsa-mir-377, hsa-mir-378, hsa-mir-378a-5p, hsa-mir-382, hsa-mir-383, hsa-mir-422a, hsa-mir-425, hsa-miR-455-3p, hsa-miR-455-5p, hsa-miR-491, hsa-mir-508, hsa-mir-508-5p, hsa-mir-512-1, hsa-mir-512-2, hsa-miR-515-3p, hsa-mir-519e, hsa-miR-520a, hsa-mir-543, hsa-mir-545, hsa-mir-549, hsa-mir-556, and hsa-miR-568, hsa-mir-620, hsa-mir-643, hsa-mir-654-3p, hsa-miR-7a-g, hsa-mir-765, hsa-mir-871, hsa-mir-888, hsa-mir-888-3p, hsa-mir-92b, hsa-mir-93, hsa-mir-96, and hsa-mir-99a. In certain embodiments of all of the above aspects, the miRNA agent is an isolated miRNA that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of a miRNA listed above. In certain embodiments of all of the above aspects, the miRNA agent is a seed sequence of a miRNA listed above.

In certain embodiments of all of the above aspects, the miRNA agent is an isolated miRNA selected from the group consisting of the 536 miRNAs set forth in Table A. In certain embodiments of all of the above aspects, the miRNA agent is an isolated miRNA that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of a miRNA listed in Table A. In certain embodiments of all of the above aspects, the miRNA agent is a seed sequence of a miRNA listed in Table A.

TABLE A Adipocyte miRNAs listed in ascending order (miRBase 19 nomenclature) hsa-let-7a-3p hsa-let-7a-5p hsa-let-7b-3p hsa-let-7b-5p hsa-let-7c hsa-let-7d-3p hsa-let-7d-5p hsa-let-7e-5p hsa-let-7f-1-3p hsa-let-7f-5p hsa-let-7g-3p hsa-let-7g-5p hsa-let-7i-3p hsa-let-7i-5p hsa-miR-1 hsa-miR-100-5p hsa-miR-101-3p hsa-miR-101-5p hsa-miR-103a-2-5p hsa-miR-103a-3p hsa-miR-103b hsa-miR-105-5p hsa-miR-106a-5p hsa-miR-106b-3p hsa-miR-106b-5p hsa-miR-107 hsa-miR-10a-3p hsa-miR-10a-5p hsa-miR-10b-3p hsa-miR-10b-5p hsa-miR-1179 hsa-miR-1185-5p hsa-miR-1208 hsa-miR-122-5p hsa-miR-1227-3p hsa-miR-1228-5p hsa-miR-1229-3p hsa-miR-124-3p hsa-miR-125a-3p hsa-miR-125a-5p hsa-miR-125b-1-3p hsa-miR-125b-2-3p hsa-miR-125b-5p hsa-miR-126-3p hsa-miR-126-5p hsa-miR-1260a hsa-miR-1260b hsa-miR-1268a hsa-miR-127-3p hsa-miR-127-5p hsa-miR-1271-5p hsa-miR-1273a hsa-miR-1277-3p hsa-miR-128 hsa-miR-128-2 hsa-miR-1285-3p hsa-miR-1287 hsa-miR-1288 hsa-miR-129-5p hsa-miR-1290 hsa-miR-1292-5p hsa-miR-1301 hsa-miR-1305 hsa-mir-1307-3p hsa-miR-130a-3p hsa-miR-130b-3p hsa-miR-130b-5p hsa-miR-132-3p hsa-miR-132-5p hsa-miR-1323 hsa-miR-133a hsa-miR-133b hsa-miR-134 hsa-miR-135a-5p hsa-miR-135b-5p hsa-miR-136-3p hsa-miR-136-5p hsa-miR-137 hsa-miR-138-1-3p hsa-miR-138-5p hsa-miR-139-3p hsa-miR-139-5p hsa-miR-140-3p hsa-miR-140-5p hsa-miR-141-3p hsa-miR-142-3p hsa-miR-142-5p hsa-miR-143-3p hsa-miR-143-5p hsa-miR-144-3p hsa-miR-144-5p hsa-miR-145-3p hsa-miR-145-5p hsa-miR-1468 hsa-miR-146a-5p hsa-miR-146b-3p hsa-miR-146b-5p hsa-miR-147a hsa-miR-148a-3p hsa-miR-148a-5p hsa-miR-148b-3p hsa-miR-148b-5p hsa-miR-149-5p hsa-miR-150-3p hsa-miR-150-5p hsa-miR-151a-3p hsa-miR-151a-5p hsa-miR-151b hsa-miR-152 hsa-miR-153 hsa-miR-1539 hsa-miR-154-3p hsa-miR-154-5p hsa-miR-155-5p hsa-miR-15a-3p hsa-miR-15a-5p hsa-miR-15b-3p hsa-miR-15b-5p hsa-miR-16-1-3p hsa-miR-16-2-3p hsa-miR-16-5p hsa-miR-17-3p hsa-miR-17-5p hsa-miR-181a-2-3p hsa-miR-181a-3p hsa-miR-181a-5p hsa-miR-181b-5p hsa-miR-181c-3p hsa-miR-181c-5p hsa-miR-181d hsa-miR-182-5p hsa-miR-183-5p hsa-miR-184 hsa-miR-185-3p hsa-miR-185-5p hsa-miR-186-3p hsa-miR-186-5p hsa-miR-187-3p hsa-miR-188-5p hsa-miR-18a-3p hsa-miR-18a-5p hsa-miR-18b-5p hsa-miR-1909-3p hsa-miR-190a hsa-miR-190b hsa-miR-191-3p hsa-miR-191-5p hsa-miR-192-5p hsa-miR-193a-3p hsa-miR-193a-5p hsa-miR-193b-3p hsa-miR-193b-5p hsa-miR-194-5p hsa-miR-195-3p hsa-miR-195-5p hsa-miR-196a-5p hsa-miR-196b-5p hsa-miR-197-3p hsa-miR-198 hsa-miR-199a-3p hsa-miR-199a-5p hsa-miR-199b-3p hsa-miR-199b-5p hsa-miR-19a-3p hsa-miR-19b-3p hsa-miR-200a-3p hsa-miR-200a-5p hsa-miR-200b-3p hsa-miR-200c-3p hsa-miR-202-3p hsa-miR-203a hsa-miR-204-5p hsa-miR-205-5p hsa-miR-206 hsa-miR-20a-3p hsa-miR-20a-5p hsa-miR-20b-5p hsa-miR-21-3p hsa-miR-21-5p hsa-miR-210 hsa-miR-211-5p hsa-miR-2110 hsa-miR-212-3p hsa-miR-214-3p hsa-miR-214-5p hsa-miR-215 hsa-miR-216a-5p hsa-miR-217 hsa-miR-218-5p hsa-miR-219-1-3p hsa-miR-219-5p hsa-miR-22-3p hsa-miR-22-5p hsa-miR-221-3p hsa-miR-221-5p hsa-miR-222-3p hsa-miR-222-5p hsa-miR-223-3p hsa-miR-223-5p hsa-miR-224-3p hsa-miR-224-5p hsa-miR-2355-3p hsa-miR-23a-3p hsa-miR-23b-3p hsa-miR-23b-5p hsa-miR-24-1-5p hsa-miR-24-2-5p hsa-miR-24-3p hsa-miR-25-3p hsa-miR-26a-2-3p hsa-miR-26a-5p hsa-miR-26b-3p hsa-miR-26b-5p hsa-miR-27a-3p hsa-miR-27a-5p hsa-miR-27b-3p hsa-miR-27b-5p hsa-miR-28-3p hsa-miR-28-5p hsa-miR-296-5p hsa-miR-297 hsa-miR-298 hsa-miR-299-3p hsa-miR-299-5p hsa-miR-29a-3p hsa-miR-29a-5p hsa-miR-29b-1-5p hsa-miR-29b-2-5p hsa-miR-29b-3p hsa-miR-29c-3p hsa-miR-29c-5p hsa-miR-301a-3p hsa-miR-301b hsa-miR-302a-5p hsa-miR-302b-5p hsa-miR-302c-5p hsa-miR-302d-3p hsa-miR-3065-3p hsa-miR-3065-5p hsa-miR-3074-3p hsa-miR-3074-5p hsa-miR-30a-3p hsa-miR-30a-5p hsa-miR-30b-3p hsa-miR-30b-5p hsa-miR-30c-1-3p hsa-miR-30c-2-3p hsa-miR-30c-5p hsa-miR-30d-3p hsa-miR-30d-5p hsa-miR-30e-3p hsa-miR-30e-5p hsa-miR-31-3p hsa-miR-31-5p hsa-miR-3120-3p hsa-miR-3120-5p hsa-miR-3184-5p hsa-miR-32-3p hsa-miR-32-5p hsa-miR-320a hsa-miR-320b hsa-miR-320c hsa-miR-323a-3p hsa-miR-324-3p hsa-miR-324-5p hsa-miR-325 hsa-miR-326 hsa-miR-328 hsa-miR-329 hsa-miR-330-3p hsa-miR-330-5p hsa-miR-331-3p hsa-miR-331-5p hsa-miR-335-3p hsa-miR-335-5p hsa-miR-337-3p hsa-miR-337-5p hsa-miR-338-3p hsa-miR-338-5p hsa-miR-339-3p hsa-miR-339-5p hsa-miR-33a-5p hsa-miR-33b-5p hsa-miR-340-3p hsa-miR-340-5p hsa-miR-342-3p hsa-miR-342-5p hsa-miR-345-5p hsa-miR-346 hsa-miR-34a-5p hsa-miR-34b-3p hsa-miR-34b-5p hsa-miR-34c-5p hsa-miR-3545-5p hsa-miR-3591-3p hsa-miR-361-3p hsa-miR-361-5p hsa-miR-3613-5p hsa-miR-3615 hsa-miR-362-3p hsa-miR-362-5p hsa-miR-363-3p hsa-miR-363-5p hsa-mir-365a-3p hsa-mir-3653 hsa-miR-3656 hsa-miR-365a-3p hsa-miR-365a-5p hsa-miR-367-3p hsa-mir-3676-3p hsa-miR-369-3p hsa-miR-369-5p hsa-miR-370 hsa-miR-371a-3p hsa-miR-373-3p hsa-miR-373-5p hsa-miR-374a-3p hsa-miR-374a-5p hsa-miR-374b-3p hsa-miR-374b-5p hsa-miR-375 hsa-mir-376a-2-5p hsa-miR-376a-3p hsa-miR-376a-5p hsa-miR-376b-3p hsa-miR-376c-3p hsa-miR-377-3p hsa-miR-378a-3p hsa-miR-378a-5p hsa-miR-378c hsa-miR-378d hsa-miR-379-5p hsa-miR-380-3p hsa-miR-381-3p hsa-miR-382-5p hsa-miR-383 hsa-miR-384 hsa-miR-3912 hsa-miR-3928 hsa-miR-409-3p hsa-miR-409-5p hsa-miR-410 hsa-miR-411-5p hsa-miR-421 hsa-miR-422a hsa-miR-422b hsa-miR-423-3p hsa-miR-423-5p hsa-miR-424-3p hsa-miR-424-5p hsa-miR-425-3p hsa-miR-425-5p hsa-miR-429 hsa-miR-431-5p hsa-miR-432-5p hsa-miR-433 hsa-miR-4421 hsa-miR-449a hsa-miR-450a-5p hsa-miR-450b-3p hsa-miR-450b-5p hsa-miR-4510 hsa-miR-4516 hsa-miR-451a hsa-miR-452-3p hsa-miR-452-5p hsa-miR-454-3p hsa-miR-454-5p hsa-miR-455-3p hsa-miR-455-5p hsa-miR-4634 hsa-miR-4732-5p hsa-miR-4792 hsa-miR-483-3p hsa-miR-483-5p hsa-miR-484 hsa-miR-485-5p hsa-miR-486-3p hsa-miR-486-5p hsa-miR-487b hsa-miR-488-3p hsa-miR-489 hsa-miR-491-3p hsa-miR-491-5p hsa-miR-492 hsa-miR-493-3p hsa-miR-493-5p hsa-miR-494 hsa-miR-495-3p hsa-miR-497-5p hsa-miR-498 hsa-miR-499a-5p hsa-miR-500a-3p hsa-miR-501-3p hsa-miR-501-5p hsa-miR-502-3p hsa-miR-502-5p hsa-miR-503-5p hsa-miR-504 hsa-miR-505-3p hsa-miR-505-5p hsa-miR-506-3p hsa-miR-509-3p hsa-miR-511 hsa-miR-513a-3p hsa-miR-513a-5p hsa-miR-513b hsa-miR-514a-3p hsa-miR-515-3p hsa-miR-516b-3p hsa-miR-516b-5p hsa-miR-518b hsa-miR-518e-3p hsa-miR-518e-5p hsa-miR-518f-3p hsa-miR-519a-5p hsa-miR-519b-5p hsa-miR-519c-3p hsa-miR-519c-5p hsa-miR-519d hsa-miR-520c-3p hsa-miR-520e hsa-miR-520f hsa-miR-520g hsa-miR-520h hsa-miR-521 hsa-miR-522-5p hsa-miR-523-5p hsa-miR-525-3p hsa-miR-532-3p hsa-miR-532-5p hsa-miR-539-5p hsa-miR-542-3p hsa-miR-542-5p hsa-miR-545-3p hsa-miR-545-5p hsa-miR-548d-3p hsa-miR-548e hsa-miR-548i hsa-miR-548m hsa-miR-550a-5p hsa-miR-551b-3p hsa-miR-552 hsa-miR-553 hsa-miR-554 hsa-miR-557 hsa-miR-563 hsa-miR-564 hsa-miR-567 hsa-miR-569 hsa-miR-570-3p hsa-miR-572 hsa-miR-574-3p hsa-miR-574-5p hsa-miR-575 hsa-miR-576-3p hsa-miR-576-5p hsa-miR-582-3p hsa-miR-582-5p hsa-miR-583 hsa-miR-584-5p hsa-miR-585 hsa-miR-586 hsa-miR-589-5p hsa-miR-590-3p hsa-miR-590-5p hsa-miR-595 hsa-miR-598 hsa-miR-601 hsa-miR-602 hsa-miR-603 hsa-miR-605 hsa-miR-606 hsa-miR-609 hsa-miR-611 hsa-miR-615-3p hsa-miR-619 hsa-miR-625-5p hsa-miR-627 hsa-miR-628-3p hsa-miR-628-5p hsa-miR-629-3p hsa-miR-629-5p hsa-miR-630 hsa-miR-636 hsa-miR-638 hsa-miR-639 hsa-miR-641 hsa-miR-642a-3p hsa-miR-642a-5p hsa-miR-646 hsa-miR-649 hsa-miR-651 hsa-miR-652-3p hsa-miR-653 hsa-miR-654-3p hsa-miR-659-3p hsa-miR-660-5p hsa-miR-663a hsa-miR-664a-3p hsa-miR-664a-5p hsa-miR-668 hsa-miR-671-5p hsa-miR-675-3p hsa-miR-675-5p hsa-miR-7-2-3p hsa-miR-7-5p hsa-miR-708-3p hsa-miR-708-5p hsa-miR-718 hsa-miR-744-5p hsa-miR-765 hsa-miR-769-5p hsa-miR-770-5p hsa-miR-874 hsa-miR-885-3p hsa-miR-887 hsa-miR-889 hsa-miR-890 hsa-miR-891a hsa-miR-891b hsa-miR-9-5p hsa-miR-92a-3p hsa-miR-92b-3p hsa-miR-93-3p hsa-miR-93-5p hsa-miR-935 hsa-miR-942 hsa-miR-95 hsa-miR-96-3p hsa-miR-96-5p hsa-miR-98-5p hsa-miR-99a-3p hsa-miR-99a-5p hsa-miR-99b-3p hsa-miR-99b-5p

In certain embodiments of all of the above aspects, the miRNA agent is a miRNA selected from the group consisting of the isolated miRNAs set forth in Table 7. In certain embodiments of all of the above aspects, the miRNA agent is an isolated miRNA that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of a miRNA listed in Table 8. In certain embodiments of all of the above aspects, the miRNA agent is a seed sequence of a miRNA listed in Table 8.

In certain embodiments of all of the above aspects, the miRNA agent is an agomir or antagomir of a miRNA selected from the group consisting of the miRNAs set forth in Table A.

In certain embodiments of all of the above aspects, the miRNA agent is an agomir or antagomir of a miRNA selected from the group consisting of hsa-miR-1-1, hsa-miR-1-2, miR-19a-b, hsa-miR-105, hsa-miR-1283, hsa-mir-129, hsa-miR-133a-1, hsa-miR-133a-2, hsa-miR-143, hsa-mir-143-5p, hsa-mir-147, hsa-mir-149, hsa-mir-199a, hsa-mir-199b, hsa-mir-200c, hsa-mir-204, hsa-mir-205, hsa-miR-206, hsa-mir-21, hsa-mir-211, hsa-mir-218, hsa-mir-218-1, hsa-mir-218-2, hsa-mir-219-2, hsa-mir-219-2-3p, hsa-mir-22, hsa-mir-22-3p, hsa-mir-22-5p, hsa-mir-24-2, hsa-miR-30a-e, hsa-miR-3177-5p, hsa-mir-325, hsa-mir-331, hsa-mir-331-5p, hsa-miR-3613-3p, hsa-mir-362, hsa-mir-362-5p, hsa-miR-3658, hsa-mir-367, hsa-mir-371, hsa-mir-371-5p, hsa-mir-377, hsa-mir-378, hsa-mir-378a-5p, hsa-mir-382, hsa-mir-383, hsa-mir-422a, hsa-mir-425, hsa-miR-455-3p, hsa-miR-455-5p, hsa-miR-491, hsa-mir-508, hsa-mir-508-5p, hsa-mir-512-1, hsa-mir-512-2, hsa-miR-515-3p, hsa-mir-519e, hsa-miR-520a, hsa-mir-543, hsa-mir-545, hsa-mir-549, hsa-mir-556, and hsa-miR-568, hsa-mir-620, hsa-mir-643, hsa-mir-654-3p, hsa-miR-7a-g, hsa-mir-765, hsa-mir-871, hsa-mir-888, hsa-mir-888-3p, hsa-mir-92b, hsa-mir-93, hsa-mir-96, and hsa-mir-99a.

In certain embodiments of all of the above aspects, the miRNA agent is an agomir or antagomir of a miRNA selected from the group consisting of the miRNAs set forth in Table 7.

In certain embodiments of all of the above aspects, the miRNA agent is an antagomir of a miRNA selected from the group consisting of hsa-miR-19b-2-5p, hsa-miR-21-5p, hsa-miR-130b-5p, hsa-miR-211, hsa-miR-325, hsa-miR-382-3p/5p, hsa-miR-543, hsa-miR-515-3p, and hsa-miR-545.

In certain embodiments of all of the above aspects, the miRNA agent is an antagomir of a miRNA selected from the group consisting of hsa-miR-331-5p, hsa-miR-552, hsa-miR-620, and hsa-miR-1179.

In certain embodiments of all of the above aspects, the miRNA agent is linked to a targeting moiety (e.g., an aptamer). In one embodiment, the targeting moiety delivers the miRNA agent to a specific cell type or tissue.

In certain embodiments of all of the above aspects, the miRNA agent directly binds to the mRNA or promoter region of at least one mitochondrial uncoupler.

In certain embodiments of all of the above aspects, the miRNA agent directly binds to the 5′UTR or coding sequence of the mRNA of at least one mitochondrial uncoupler.

In certain embodiments of all of the above aspects, the miRNA agent directly binds to the 3′UTR of the mRNA of at least one mitochondrial uncoupler.

In certain embodiments of all of the above aspects, the miRNA agent modulates the activity of an activator or repressor of a mitochondrial uncoupling protein. In one embodiment, the miRNA agent directly binds to the mRNA or promoter region of the activator or repressor. In one embodiment, the miRNA agent directly binds to the 5′UTR or coding sequence of the mRNA of the activator or repressor. In one embodiment, the miRNA agent directly binds to the 3′UTR of the mRNA of the activator or repressor. In one embodiment, the activator or repressor is selected from the group listed in Table 1.

In certain embodiments of all of the above aspects, the mRNA or protein expression of the mitochondrial uncoupling protein is upregulated.

In certain embodiments of all of the above aspects, the mitochondrial uncoupling activity of the mitochondrial uncoupling protein is upregulated.

In another aspect, the invention provides a method of screening for a miRNA agent that modulates thermogenesis, the method generally comprising: providing an indicator cell; contacting the indicator cell with a test miRNA agent; and determining the cellular activity of at least one thermogenic regulator in the indicator cell in the presence and absence of the miRNA agent, wherein a change in the activity of the thermogenic regulator in the presence of the test miRNA agent identifies the test miRNA agent as a miRNA agent that modulates thermogenesis. The indicator cell can be a mammalian cell. In certain embodiments, the indicator cell is a human cell comprising at least a portion of a human genome.

In certain embodiments, the cell is a pre-adipocyte, adipocyte, adipose tissue derived mesenchymal stem cell, hepatocyte, myocyte, or a precursor thereof.

In certain embodiments, the cellular activity of the thermogenic regulator determined in the method is the mRNA expression level, protein expression level or mitochondrial uncoupling activity of the thermogenic regulator.

In certain embodiments, the test miRNA agent increases the activity of the thermogenic regulator compared to the level of activity of the thermogenic regulator in the absence of the test miRNA agent.

In certain embodiments, the thermogenic regulator is UCP1 or UCP2.

In another aspect, the invention provides an agomir or antagomir that modulates the activity of at least one thermogenic regulator in a cell.

In certain embodiments, the agomir or antagomir is an agomir or antagomir of a miRNA selected from the group consisting of the miRNAs set forth in Table 8.

In certain embodiments, the agomir or antagomir is an agomir or antagomir of a miRNA selected from the group consisting of the miRNAs set forth in Table A.

In certain embodiments, the agomir or antagomir is an agomir or antagomir of a miRNA selected from the group consisting of hsa-miR-1-1, hsa-miR-1-2, miR-19a-b, hsa-miR-105, hsa-miR-1283, hsa-mir-129, hsa-miR-133a-1, hsa-miR-133a-2, hsa-miR-143, hsa-mir-143-5p, hsa-mir-147, hsa-mir-149, hsa-mir-199a, hsa-mir-199b, hsa-mir-200c, hsa-mir-204, hsa-mir-205, hsa-miR-206, hsa-mir-21, hsa-mir-211, hsa-mir-218, hsa-mir-218-1, hsa-mir-218-2, hsa-mir-219-2, hsa-mir-219-2-3p, hsa-mir-22, hsa-mir-22-3p, hsa-mir-22-5p, hsa-mir-24-2, hsa-miR-30a-e, hsa-miR-3177-5p, hsa-mir-325, hsa-mir-331, hsa-mir-331-5p, hsa-miR-3613-3p, hsa-mir-362, hsa-mir-362-5p, hsa-miR-3658, hsa-mir-367, hsa-mir-371, hsa-mir-371-5p, hsa-mir-377, hsa-mir-378, hsa-mir-378a-5p, hsa-mir-382, hsa-mir-383, hsa-mir-422a, hsa-mir-425, hsa-miR-455-3p, hsa-miR-455-5p, hsa-miR-491, hsa-mir-508, hsa-mir-508-5p, hsa-mir-512-1, hsa-mir-512-2, hsa-miR-515-3p, hsa-mir-519e, hsa-miR-520a, hsa-mir-543, hsa-mir-545, hsa-mir-549, hsa-mir-556, and hsa-miR-568, hsa-mir-620, hsa-mir-643, hsa-mir-654-3p, hsa-miR-7a-g, hsa-mir-765, hsa-mir-871, hsa-mir-888, hsa-mir-888-3p, hsa-mir-92b, hsa-mir-93, hsa-mir-96, and hsa-mir-99a.

In certain embodiments, the agomir or antagomir is an antagomir of a miRNA selected from the group consisting of hsa-miR-19b-2-5p, ha-miR-21-5p, hsa-miR-130b-5p, hsa-miR-211, hsa-miR-325, hsa-miR-382-3p/5p, hsa-miR-543, hsa-miR-515-3p, and hsa-miR-545.

In certain embodiments, the agomir or antagomir is an antagomir of a miRNA selected from the group consisting of hsa-miR-331-5p, hsa-miR-552, hsa-miR-620, and hsa-miR-1179.

In certain embodiments, the agomir or antagomir is linked to a targeting moiety.

In certain embodiments, the targeting moiety is an aptamer.

In certain embodiments, the targeting moiety delivers the agomir or antagomir to a specific cell type or tissue.

In certain embodiments, the agomir or antagomir directly binds to the mRNA or promoter region of at least one mitochondrial uncoupler.

In certain embodiments, the agomir or antagomir directly binds to the 5′UTR or coding sequence of the mRNA of at least one mitochondrial uncoupler.

In certain embodiments, the agomir or antagomir directly binds to the 3′UTR of the mRNA of at least one mitochondrial uncoupler.

In certain embodiments, the agomir or antagomir modulates the activity of an activator or repressor of a mitochondrial uncoupling protein.

In certain embodiments, the activator or repressor is selected from the group listed in Table 1.

In certain embodiments, the agomir or antagomir directly binds to the mRNA or promoter region of the activator or repressor.

In certain embodiments, the agomir or antagomir directly binds to the 5′UTR or coding sequence of the mRNA of the activator or repressor. In other embodiments, the agomir or antagomir directly binds to the 3′UTR of the mRNA of the activator or repressor.

The disclosure also provides a pharmaceutical composition comprising two or more miRNAs selected from hsa-let-7a agomir, hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-1 antagomir, hsa-miR-19b agomir, hsa-miR-19b antagomir, hsa-miR-30b agomir and hsa-miR-30b antagomir. In certain embodiments the pharmaceutical composition also includes a pharmaceutically acceptable excipient. In certain embodiments, the two or more miRNAs are expressed from a recombinant vector. The recombinant vector can be selected from DNA plasmids, viral vectors and DNA minicircles.

The disclosure also provides a method of inducing pre-adipocytes to differentiate initially into white adipocytes and subsequently into brown adipocytes comprising administering to a population of pre-adipocytes one or more miRNAs selected from hsa-let-7a agomir, hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-1 antagomir, hsa-miR-19b agomir, hsa-miR-19b antagomir, hsa-miR-30b agomir and hsa-miR-30b antagomir. The one or more miRNAs can also be selected from hsa-let-7a agomir, hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-1 antagomir, hsa-miR-19b agomir, hsa-miR-19b antagomir, hsa-miR-30b agomir and hsa-miR-30b antagomir. In certain embodiments, the induction of pre-adipocytes to differentiate into adipocytes is greater than the differentiation of pre-adipocytes to adipocytes when pre-adipocytes are exposed to 100 mM rosiglitazone for two days followed by maintenance medium. In certain embodiments, the adipocytes are brown adipocytes. In other embodiments, the adipocytes are white adipocytes. Additional criteria for differentiation can be found in the Examples, below.

The disclosure also provides a method for decreasing the lipid content of adipocytes comprising administering to a population of adipocytes one or more miRNAs selected from the group consisting of hsa-let-7a agomir, hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-1 antagomir, hsa-miR-19b agomir, hsa-miR-19b antagomir, hsa-miR-30b agomir and hsa-miR-30b antagomir. In certain embodiments, the lipid content of the adipocytes is less than the lipid content of adipocytes exposed to 100 nM rosiglitazone for two days followed by maintenance medium or less than the fat content of adipocytes exposed to 100 nM rosiglitazone for the duration of culture. The duration of culture can be 8-16, 10-14 or 14 days. The duration of culture can also be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days. Additional criteria for lipid content of adipocytes can be found in the Examples, below.

The disclosure also provides a method for increasing insulin sensitivity in a subject in need thereof comprising administering the subject one or more miRNAs selected from the group consisting of hsa-let-7a agomir, hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-1 antagomir, hsa-miR-19b agomir, hsa-miR-19b antagomir, hsa-miR-30b agomir and hsa-miR-30b antagomir.

In certain embodiments, the subject is a mammal.

The disclosure also provides a method of increasing expression or activity of one or more uncoupling proteins in a cell comprising administering to the cell one or more, two or more, or three or more miRNAs selected from the group consisting of hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-19b agomir and hsa-miR-30b agomir. In certain embodiments, the cell is selected from the group consisting of a brown adipocyte, a white adipocyte, a subcutaneous adipocyte, a liver cell or a muscle cell. In other embodiments, the one or more uncoupling proteins include UCP1 or UCP2. In certain embodiments, the method is an ex vivo method. In other embodiments, the method is an in vivo method. In certain embodiments, the method involves selecting a subject (e.g., a human) in need of increasing the level of expression or activity of one or more uncoupling proteins (e.g., UCP1, UCP2). In some embodiments, the subject has, or is at risk of developing, obesity. In certain embodiments, the subject has, or is at risk of developing, diabetes. In certain embodiments, the method further comprises determining the expression level (mRNA or protein) or activity of the one or more uncoupling proteins.

The disclosure also provides a method of causing fat loss in a subject in need thereof comprising administering the subject one or more miRNAs selected from the group consisting of hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-19b agomir and hsa-miR-30b agomir. In certain embodiments, the subject is a mammal. In other embodiments, the mammal is a human.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are schematic representations of the interactions of 83 thermogenic regulators determined using the STRING 9.0 database.

FIG. 2A is a schematic representation of the interaction of 83 thermogenic regulators determined using the Ingenuity Pathway Analysis Software program.

FIG. 2B is a schematic representation of the interaction of 83 thermogenic regulators determined using the Reactome Functional Interaction Network program.

FIG. 3 is a schematic representation of the overlap between the visual inspection and alignment of nucleotide sequences set forth herein, and the results from multiple miRNA prediction programs predicting miRNA binding sites in the 5′UTR, promoter region, coding sequence and 3′UTR of the human UCP1 gene.

FIG. 4 is a schematic representation of the overlap of results from multiple miRNA prediction programs predicting miRNA binding sites in the 5′UTR, promoter region, coding sequence and 3′UTR of the genes of 83 thermogenic regulators.

FIG. 5 is a schematic representation of oxidative phosphorylation in mitochondria, illustrating the uncoupling of oxidative phosphorylation from ATP synthesis by UCP1 to generate heat.

FIG. 6 depicts the transcriptional control of UCP1 by other exemplary thermogenic regulators.

FIG. 7 depicts exemplary positive (A) and negative (B) transcriptional regulators of the UCP1 gene.

FIG. 8A depicts the location of various regulatory elements in reference to the transcription start site in the 15,910 base pair (bp) human UCP1 gene sequence (NCBI Reference Sequence: gi|237858805|ref|NG_012139.1| Homo sapiens uncoupling protein 1 (mitochondrial, proton carrier) (UCP1), RefSeqGene on chromosome 4).

FIG. 8B depicts the location of various regulatory elements in reference to the transcription start site in the 15,174 bp of the human UCP2 gene (ENSG00000175567), including 5,000 bp 5′UTR and 2,000 bp 3′UTR on chromosome 11.

FIG. 9 is a bar graph showing relative fluorescence in unlabeled cells or cells transfected with a Dy547 labeled non-targeting miRIDIAN mimic and hairpin inhibitor.

FIG. 10A is a bar graph showing the reduction of GAPDH expression in cells transfected with siRNA control and a GAPDH siRNA 4 days after transfection.

FIG. 10B is a bar graph showing the reduction of GAPDH expression in cells transfected with siRNA control and a GAPDH siRNA 12 days after transfection.

FIG. 11A is a light micrograph of preadipocytes stained with Oil Red 0 cultured for 2 weeks in maintenance medium without rosiglitazone.

FIG. 11B is a light micrograph of preadipocytes stained with Oil Red 0 cultured in the presence of insulin, triiodothyronine, dexamethasone, isobutyl-methylxanthine and rosiglitazone for two days followed by maintenance medium for 12 days.

FIG. 11C is a light micrograph of preadipocytes stained with Oil Red 0 cultured in the presence of insulin, triiodothyronine, dexamethasone, isobutyl-methylxanthine and rosiglitazone throughout the experiment.

FIG. 11D is a light micrograph of preadipocytes stained with Oil Red 0 cultured in the presence of hsa-miR-30b mimic.

FIG. 11E is a light micrograph of preadipocytes stained with Oil Red 0 cultured in the presence of non targeting miRNA mimic.

FIG. 11F is a light micrograph of preadipocytes stained with Oil Red 0 cultured in the presence of non targeting miRNA inhibitor.

FIG. 12A is a bar graph showing mRNA expression of thermogenesis targets in the presence of rosiglitazone.

FIG. 12B is a bar graph showing mRNA expression of thermogenesis targets in the presence of hsa-let-7a inhibitor.

FIG. 12C is a bar graph showing mRNA expression of thermogenesis targets in the presence of hsa-miR-1 mimic.

FIG. 12D is a bar graph showing mRNA expression of thermogenesis targets in the presence of hsa-miR-19b mimic.

FIG. 12E is a bar graph showing mRNA expression of thermogenesis targets in the presence of and hsa-miR-30b mimic.

FIG. 12F is a bar graph showing mRNA expression of thermogenesis targets in untreated preadipocytes.

FIG. 13 is a bar graph showing relative fluorescence in unlabeled cells and cells transfected with a Dy547 labeled non-targeting miRIDIAN mimic or hairpin inhibitor.

FIG. 14A is a bar graph showing the reduction of GAPDH expression in cells transfected with siRNA control and a GAPDH siRNA 4 days after transfection.

FIG. 14B is a bar graph showing the reduction of GAPDH expression in cells transfected with siRNA control and a GAPDH siRNA 12 days after transfection.

FIG. 15 is a bar graph showing the amount of lipids in mature adipocytes using Nile Red Dye exposed to various miRNAs.

FIG. 16 is an M-A plot showing the mean gene expression on the x-axis and the difference between pairs in logarithmic scale on the y-axis.

FIG. 17 is a schematic showing a Venn Diagram showing that the numbers of genes significantly upregulated in the presence of the miRNA analogs hsa-let-7a inhibitor, hsa-miR-1 mimic, hsa-miR-19b mimic and hsa-miR-30b mimic were respectively 305, 247, 255 and 267. A set of 127 genes was commonly upregulated by the listed miRNA analogs.

FIG. 18 is a schematic showing a Venn diagram showing that the numbers of genes significantly downpregulated in the presence of the miRNA analogs hsa-let-7a inhibitor, hsa-miR-1 mimic, hsa-miR-19b mimic and hsa-miR-30b mimic were respectively 143, 177, 115 and 165. A set of 60 genes that was commonly downregulated by the listed miRNA analogs.

FIG. 19 is a bar graph showing the amounts of RNA extracted from mature adipocytes exposed to various transfecting agents.

FIG. 20 is a bar graph showing reduction of GAPDH expression in mature adipocytes transfected with a GAPDH-specific miRNA mimic using various transfecting agents.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application, including definitions, will control.

As used herein, the term “miRNA agent” refers to an oligonucleotide or oligonucleotide mimetic that directly or indirectly modulates the activity of a thermogenic regulator (e.g., a mitochondrial uncoupler or an activator or repressor thereof). miRNA agents can act on a target gene or on a target miRNA.

As used herein, the term “miRNA” refers to a single-stranded RNA molecule (or a synthetic derivative thereof), which is capable of binding to a target gene (either the mRNA or the DNA) and regulating expression of that gene. In certain embodiments, the miRNA is naturally expressed in an organism.

As used herein, the term “seed sequence” refers to a 6-8 nucleotide (nt) long substring within the first 8 nt at the 5′-end of the miRNA (i.e., seed sequence) that is an important determinant of target specificity.

As used herein, the term “agomir” refers to a synthetic oligonucleotide or oligonucleotide mimetic that functionally mimics a miRNA. An agomir can be an oligonucleotide with the same or similar nucleic acid sequence to a miRNA or a portion of a miRNA. In certain embodiments, the agomir has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide differences from the miRNA that it mimics. Further, agomirs can have the same length, a longer length or a shorter length than the miRNA that it mimics. In certain embodiments, the agomir has the same sequence as 6-8 nucleotides at the 5′ end of the miRNA it mimics. In other embodiments, an agomir can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In other embodiments, an agomir can be 5-10, 6-8, 10-20, 10-15 or 5-500 nucleotides in length. In certain embodiments, agomirs include any of the sequences shown in Table A. These chemically modified synthetic RNA duplexes include a guide strand that is identical or substantially identical to the miRNA of interest to allow efficient loading into the miRISC complex, whereas the passenger strand is chemically modified to prevent its loading to the Argonaute protein in the miRISC complex (Thorsen S B et al., Cancer J., 18(3):275-284 (2012); Broderick J A et al., Gene Ther., 18(12):1104-1110 (2011).

As used herein, the term “antagomir” refers to a synthetic oligonucleotide or oligonucleotide mimetic having complementarity to a specific microRNA, and which inhibits the activity of that miRNA. In certain embodiments, the antagomir has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide differences from the miRNA that it inhibits. Further, antagomirs can have the same length, a longer length or a shorter length than the miRNA that it inhibits. In certain embodiments, the antagomir hybridizes to 6-8 nucleotides at the 5′ end of the miRNA it inhibits. In other embodiments, an antagomir can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides in length. In other embodiments, an antagomir can be 5-10, 6-8, 10-20, 10-15 or 5-500 nucleotides in length. In certain embodiments, antagomirs include nucleotides that are complementary to any of the sequences shown in Table A. The antagomirs are synthetic reverse complements that tightly bind to and inactivate a specific miRNA. Various chemical modifications are used to improve nuclease resistance and binding affinity. The most commonly used modifications to increase potency include various 2′ sugar modifications, such as 2′-O-Me, 2′-O-methoxyethyl (2′-MOE), or 2′-fluoro(2′-F). The nucleic acid structure of the miRNA can also be modified into a locked nucleic acid (LNA) with a methylene bridge between the 2′ oxygen and the 4′ carbon to lock the ribose in the 3′-endo (North) conformation in the A-type conformation of nucleic acids (Lennox K A et al. Gene Ther. December 2011; 18(12):1111-1120; Bader A G et al. Gene Ther. December 2011; 18(12):1121-1126). This modification significantly increases both target specificity and hybridization properties of the molecules.

As used herein, the term “aptamir” refers to the combination of an aptamer (oligonucleic acid or peptide molecule that bind to a specific target molecule) and an agomir or antagomir as defined above, which allows cell or tissue-specific delivery of the miRNA agents.

As used herein, the term “interfering RNA” refers to any double stranded or single stranded RNA sequence capable of inhibiting or down-regulating gene expression by mediating

RNA interference. Interfering RNAs include, but are not limited to, small interfering RNA (“siRNA”) and small hairpin RNA (“shRNA”). “RNA interference” refers to the selective degradation of a sequence-compatible messenger RNA transcript.

As used herein, the term “small interfering RNA” or “siRNA” refers to any small RNA molecule capable of inhibiting or down regulating gene expression by mediating RNA interference in a sequence specific manner. The small RNA can be, for example, about 16 to 21 nucleotides long.

As used herein, the term “shRNA” (small hairpin RNA) refers to an RNA molecule comprising an antisense region, a loop portion and a sense region, wherein the sense region has complementary nucleotides that base pair with the antisense region to form a duplex stem. Following post-transcriptional processing, the small hairpin RNA is converted into a small interfering RNA (siRNA) by a cleavage event mediated by the enzyme Dicer, which is a member of the RNase III family.

As used herein, the term “antisense oligonucleotide” refers to a synthetic oligonucleotide or oligonucleotide mimetic that is complementary to a DNA or mRNA sequence (e.g., a miRNA).

As used herein, the term “miR-mask” refers to a single stranded antisense oligonucleotide that is complementary to a miRNA binding site in a target mRNA, and that serves to inhibit the binding of miRNA to the mRNA binding site. See, e.g., Xiao, et al. “Novel approaches for gene-specific interference via manipulating actions of microRNAs: examination on the pacemaker channel genes HCN2 and HCN4,” Journal of Cellular Physiology, vol. 212, no. 2, pp. 285-292, 2007, which is incorporated herein in its entirety.

As used herein, the term “miRNA sponge” refers to a synthetic nucleic acid (e.g. a mRNA transcript) that contains multiple tandem-binding sites for a miRNA of interest, and that serves to titrate out the endogenous miRNA of interest, thus inhibiting the binding of the miRNA of interest to its endogenous targets. See, e.g., Ebert et al., “MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells,” Nature Methods, vol. 4, no. 9, pp. 721-726, 2007, which is incorporated herein in its entirety.

As used herein, the term “respiratory chain uncoupling” refers to the dissipation of the mitochondrial inner membrane proton gradient, thereby preventing the synthesis of ATP in the mitochondrion by oxidative phosphorylation.

As used herein, the term “mitochondrial uncoupler” refers to a protein (or the encoding nucleic acid) that can dissipate of the mitochondrial inner membrane proton gradient, thereby preventing the synthesis of ATP in the mitochondrion by oxidative phosphorylation. Exemplary mitochondrial uncouplers include UCP1 and UCP2.

As used herein, the terms “activator” or “repressor” of a mitochondrial uncoupler refers to a protein that serves to upregulate or downregulate, respectively, an activity of a mitochondrial uncoupler.

As used herein, the term “thermogenic regulator” refers to a protein (or the encoding nucleic acid) that regulates thermogenesis either directly or indirectly. The term encompasses mitochondrial uncouplers, and also activators and repressors of mitochondrial uncouplers. Exemplary thermogenic regulators are set forth in Table 1 herein.

As used herein, the term “modulate” refers to increasing or decreasing a parameter. For example, to modulate the activity of a protein that protein's activity could be increased or decreased.

As used herein, the term “activity” of mitochondrial uncoupler or thermogenic regulator refers to any measurable biological activity including, without limitation, mRNA expression, protein expression, or respiratory chain uncoupling.

The “effective amount” of the miRNA agent composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans.

In certain embodiments, this physiological condition is obesity.

A “subject” is a vertebrate, including any member of the class Mammalia, including humans, domestic and farm animals, zoo, sports or pet animals, such as mouse, rabbit, pig, sheep, goat, cattle and higher primates.

The term “mammal” refers to any species that is a member of the class mammalia, including rodents, primates, dogs, cats, camelids and ungulates. The term “rodent” refers to any species that is a member of the order rodentia including mice, rats, hamsters, gerbils and rabbits. The term “primate” refers to any species that is a member of the order primates, including monkeys, apes and humans. The term “camelids” refers to any species that is a member of the family camelidae including camels and llamas. The term “ungulates” refers to any species that is a member of the superorder ungulata including cattle, horses and camelids. According to some embodiments, the mammal is a human.

“Treatment”, or “treating” as used herein, is defined as the application or administration of a therapeutic agent (e.g., a miRNA agent or vector or transgene encoding same) to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient, who has the disease or disorder, a symptom of disease or disorder or a predisposition toward a disease or disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect the disease or disorder, the symptoms of the disease or disorder, or the predisposition toward disease.

“Pharmacogenomics”, as used herein, refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drugs in clinical development and on the market. More specifically, the term refers to the study of how a patient's genes determine his or her response to a drug (e.g., a patient's “drug response phenotype”, or “drug response genotype”).

The “effective amount” of the miRNA agent composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans.

II. Thermogenesis and Obesity

In certain embodiments, the invention provides methods for modulating thermogenesis. These methods generally involve contacting cells or tissue with a miRNA agent that modulates activity of at least one mitochondrial uncoupler (e.g., UCP1 and/or UCP2). Such methods and compositions are particularly useful for treating obesity.

Mammalian adipocytes can be categorized into two major categories based on their functional profiles: 1) energy-storing and releasing, lipid-filled white adipocytes (WAT) and; 2) energy-expending and heat producing, mitochondria-rich brown adipocytes (BAT). Until recently, it was believed that BAT underwent rapid involution in early childhood, leaving only vestigial amounts in adults. However, positron-emission tomography (PET) studies performed in humans with the tracer 18F-fluorodeoxyglucose (18F-FDG) demonstrated that: 1) multiple depots of BAT are still present in the cervical, supraclavicular, axillary and paravertebral regions in adult subjects; 2) BAT in adult humans can be rapidly activated by exposure to cold temperatures; 3) there is an inverse correlation between the activity of BAT and age, body-mass index (BMI), the percentage of body fat, fasting plasma glucose level, beta-blocker use and outdoor temperature; and 4) BAT expansion may drive the weight loss associated with catecholamine-producing phaeochromocytomas, whereas beta3-adrenoreceptor polymorphisms leading to a reduction in receptor function have been linked to weight gain and early onset type 2 diabetes mellitus.

Although WAT and BAT are derived from mesenchymal stem cells, they have distinct lineages, with Myf5 (Myogenic Regulatory Factor 5) (shared with skeletal myocyte progenitors), PGC-lalpha and PRDM16 (PR-domain-containing 16) expression distinguishing the brown from white adipocyte precursors. In addition to the classic brown adipocytes, a different type of brown fat cells can be induced in tissues where WAT predominates. The termed “brite” (brown-in-white) adipocyte has been coined and the appearance of brown-like adipocytes within WAT depots is associated with improved metabolic phenotypes. Increasing BAT mass and/or activity offers a degree of protection from obesity. Heat production by BAT is 300 W/g compared to 1 W/g in all other tissues. Relatively limited amounts of BAT would be required to make significant impact on energy balance, since as little as 50 g of BAT would account for 20% of daily energy expenditure. It has been speculated that the estimated 63 g of BAT found in the supraclavicular/paracervical depot of one subject could combust the energy equivalent of 4.1 kg of WAT over 1 year.

Mitochondrial uncoupling proteins (UCP) are members of the family of mitochondrial anion carrier proteins (MACP). UCPs separate oxidative phosphorylation from ATP synthesis with energy dissipated as heat (also referred to as the “mitochondrial proton leak”). UCPs facilitate the transfer of anions from the inner to the outer mitochondrial membrane and the return transfer of protons from the outer to the inner mitochondrial membrane generating heat in the process. UCPs are the primary proteins responsible for thermogenesis and heat dissipation. Uncoupling Protein 1 (UCP1), also named thermogenin, is a BAT specific protein responsible for thermogenesis and heat dissipation. UCP2 is another Uncoupling Protein also expressed in adipocytes. UCPs are part of network of thermogenic regulator proteins (see FIG. 1). Exemplary thermogenic regulators are set forth in Table 1.

Modulation of thermogenic regulators to induce BAT differentiation and/or mitochondrial uncoupling proteins provides a method to induce thermogenesis in a subject and, hence, to treat obesity. However, chemical pharmacologic approaches cannot target these molecules, as they do not belong to the classic ‘target classes’ (kinases, ion channels, G-protein coupled receptors, etc.) that dominate the druggable space′ of traditional drug discovery. Accordingly, the invention provides novel methods and compositions for modulating these thermogenic regulators using miRNA agents.

In certain embodiments, miRNA agents are employed to upregulate the activity of a mitochondrial uncoupler (e.g., the mRNA expression level, protein expression level, or mitochondrial uncoupling activity). Upregulation of a mitochondrial uncoupler can be achieved in several ways. In one embodiment, the miRNA agent directly inhibits the activity of a naturally occurring miRNA that is responsible for downregulation of the activity (e.g., the mRNA expression level, protein expression level) of the mitochondrial uncoupler. In another embodiment, the miRNA agent upregulates the activity (e.g., the mRNA expression level or the protein expression level) of an activator of the mitochondrial uncoupler. This upregulation can be achieved, for example, by directly inhibiting the activity of a naturally occurring miRNA that is responsible for downregulation of the expression of the activator. In yet another embodiment, the miRNA agent downregulates the activity (e.g., the mRNA expression level or the protein expression level) of a repressor of the mitochondrial uncoupler. This downregulation can be achieved, for example, by directly inhibiting the expression of a repressor of a mitochondrial uncoupler using a miRNA agent.

In certain embodiments, miRNA agents are employed that are capable of modulating the activity of multiple thermogenic regulators simultaneously (Pathway-specific miRNA agents as opposed to universal miRNA agents). For example, a single miRNA, agomir or antagomir that binds to multiple thermogenic regulators can be used. This approach is particularly advantageous in that it allows for the modulation of multiple members of an entire signaling pathway using a single miRNA agent.

In certain embodiments, multiple inhibitory miRNA agents (e.g., antagomirs or miR-masks) are employed. These inhibitory miRNA agents can have the same or different miRNA targets.

III. miRNA Agents

In certain embodiments, the invention employs miRNA agents for the modulation of thermogenic regulators (e.g., mitochondrial uncouplers, such as UCP1 and/or UCP2). miRNA agents, suitable for use in the methods disclosed herein, included, without limitation, miRNA, agomirs, antagomirs, miR-masks, miRNA-sponges, siRNA (single- or double-stranded), shRNA, antisense oligonucleotides, ribozymes, or other oligonucleotide mimetics which hybridize to at least a portion of a target nucleic acid and modulate its function.

In certain embodiments, the miRNA agents are miRNA molecules or synthetic derivatives thereof (e.g., agomirs). In one particular embodiment, the miRNA agent is a miRNA. miRNAs are a class of small (e.g., 18-24 nucleotides) non-coding RNAs that exist in a variety of organisms, including mammals, and are conserved in evolution. miRNAs are processed from hairpin precursors of about 70 nucleotides which are derived from primary transcripts through sequential cleavage by the RNAse III enzymes drosha and dicer. Many miRNAs can be encoded in intergenic regions, hosted within introns of pre-mRNAs or within ncRNA genes. Many miRNAs also tend to be clustered and transcribed as polycistrons and often have similar spatial temporal expression patterns. In general, miRNAs are post-transcriptional regulators that bind to complementary sequences on a target gene (mRNA or DNA), resulting in gene silencing by, e.g., translational repression or target degradation. One miRNA can target many different genes simultaneously. Exemplary miRNA molecules for use in the disclosed methods include without limitation: hsa-miR-1-1, hsa-miR-1-2, hsa-miR-7a-g, hsa-miR-105, hsa-miR-1283, hsa-mir-129, hsa-miR-133a-1, hsa-miR-133a-2, hsa-miR-143, hsa-mir-143-5p, hsa-mir-147, hsa-mir-149, hsa-miR-19a-b, hsa-mir-199a, hsa-mir-199b, hsa-mir-200c, hsa-mir-204, hsa-mir-205, hsa-miR-206, hsa-mir-21, hsa-mir-211, hsa-mir-218, hsa-mir-218-1, hsa-mir-218-2, hsa-mir-219-2, hsa-mir-219-2-3p, hsa-mir-22, hsa-mir-22-3p, hsa-mir-22-5p, hsa-mir-24-2, hsa-miR-30a-e, hsa-miR-3177-5p, hsa-mir-325, hsa-mir-331, hsa-mir-331-5p, hsa-miR-3613-3p, hsa-mir-362, hsa-mir-362-5p, hsa-mir-367, hsa-mir-371, hsa-mir-371-5p, hsa-mir-377, hsa-mir-378, hsa-mir-378a-5p, hsa-mir-382, hsa-mir-383, hsa-miR-3658, hsa-mir-422a, hsa-mir-425, hsa-miR-455-3p, hsa-miR-455-5p, hsa-miR-491, hsa-mir-508, hsa-mir-508-5p, hsa-mir-512-1, hsa-mir-512-2, hsa-miR-515-3p, hsa-mir-519e, hsa-miR-520a, hsa-mir-543, hsa-mir-545, hsa-mir-549, hsa-mir-556, hsa-miR-568, hsa-mir-620, hsa-mir-643, hsa-mir-654-3p, hsa-mir-765, hsa-mir-871, hsa-mir-888, hsa-mir-888-3p, hsa-mir-92b, hsa-mir-93, hsa-mir-96, hsa-mir-99a. In other embodiments, exemplary miRNA molecules for use in the disclosed methods miRNA disclosed in Table A and/or Table 8, herein. In one particular embodiment, the miRNA agent is human miR-22, or a functional derivative thereof.

In another particular embodiment, the miRNA agent is an agomir. Agomirs of a particular miRNA can be identified using the screening methods disclosed herein. In one particular embodiment, the agomir is a functional mimetic of human miR-22 (Davidson B L et al., Nat Rev Genet., 12(5):329-340 (2011).

In certain embodiments, the miRNA agents are oligonucleotide or oligonucleotide mimetics that inhibit the activity of one or more miRNA. Examples of such molecules include, without limitation, antagomirs, interfering RNA, antisense oligonucleotides, ribozymes, miRNA sponges and miR-masks. In one particular embodiment, the miRNA agent is an antagomir. In general, antagomirs are chemically modified antisense oligonucleotides that bind to a target miRNA and inhibit miRNA function by preventing binding of the miRNA to its cognate gene target. Antagomirs can include any base modification known in the art. In one particular embodiment, the antagomir inhibits the activity of human miR-22 (van Rooij E et al., Circ Res., 110(3):496-507 (2012); Snead N M et al., Nucleic Acid Ther., 22(3):139-146 (2012); Czech M P et al., Nat Rev Endocrinol., 7(8):473-484 (2011).

In certain embodiments, the miRNA agents are 10 to 50 nucleotides in length. One having ordinary skill in the art will appreciate that this embodies oligonucleotides having antisense portions of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length, or any range therewithin.

In certain embodiments, the miRNA agents are chimeric oligonucleotides that contain two or more chemically distinct regions, each made up of at least one nucleotide. These oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target) and a region that is a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. Chimeric inhibitory nucleic acids of the invention may be formed as composite structures of two or more oligonucleotides, modified oligonucleotides, oligonucleosides and/or oligonucleotide mimetics as described above. Such compounds have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures comprise, but are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference in its entirety.

In certain embodiments, the miRNA agents comprise at least one nucleotide modified at the 2′ position of the sugar, most preferably a 2′-O-alkyl, 2′-O-alkyl-O-alkyl or 2′-fluoro-modified nucleotide. In other preferred embodiments, RNA modifications include 2′-fluoro, 2′-amino and 2′ O-methyl modifications on the ribose of pyrimidines, a basic residue or an inverted base at the 3′ end of the RNA. Such modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than 2′-deoxyoligonucleotides against a given target.

A number of nucleotide and nucleoside modifications have been shown to make an oligonucleotide more resistant to nuclease digestion, thereby prolonging in vivo half-life. Specific examples of modified oligonucleotides include those comprising backbones comprising, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are oligonucleotides with phosphorothioate backbones and those with heteroatom backbones, particularly CH₂—NH—O—CH₂, CH, ˜N(CH₃)˜O˜CH₂ (known as a methylene(methylimino) or MMI backbone], CH₂—O—N(CH₃)—CH₂, CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂—CH₂ backbones, wherein the native phosphodiester backbone is represented as O—P—O—CH); amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbone structures (see Summerton and Weller, U.S. Pat. No. 5,034,506); peptide nucleic acid (PNA) backbone (wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497), each of which is herein incorporated by reference in its entirety. Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3′ alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3 ‘-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3’-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321, 131; 5,399,676; 5,405,939; 5,453,496; 5,455, 233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference in its entirety. Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510); Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol, 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991, each of which is herein incorporated by reference in its entirety. Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wang et al., J. Am. Chem. Soc., 2000, 122, 8595-8602, the contents of which is incorporated herein in its entirety.

Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216, 141; 5,235,033; 5,264,562; 5, 264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference in its entirety.

In certain embodiments, miRNA agents comprise one or more substituted sugar moieties, e.g., one of the following at the 2′ position: OH, SH, SCH₃, F, OCN, OCH₃OCH₃, OCH₃O(CH₂)nCH₃, O(CH₂)nNH₂ or O(CH₂)n CH₃ where n is from 1 to about 10; Ci to CIO lower alkyl, alkoxyalkoxy, substituted lower alkyl, alkaryl or aralkyl; CI; Br; CN; CF3; OCF3; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacokinetic/pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. A preferred modification includes 2′-methoxyethoxy [2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl)] (Martin et al., Helv. Chim. Acta, 1995, 78, 486). Other preferred modifications include 2′-methoxy (2′-O—CH₃), 2′-propoxy (2′-OCH₂CH₂CH₃) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3′ position of the sugar on the 3′ terminal nucleotide and the 5′ position of 5′ terminal nucleotide. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group.

In certain embodiments, miRNA agents comprise one or more base modifications and/or substitutions. As used herein, “unmodified” or “natural” bases include adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U). Modified bases include, without limitation, bases found only infrequently or transiently in natural nucleic acids, e.g., hypoxanthine, 6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (also referred to as 5-methyl-2′ deoxycytosine and often referred to in the art as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC and gentobiosyl HMC, as well as synthetic bases, e.g., 2-aminoadenine, 2-(methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil, 2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine, N6 (6-aminohexyl)adenine and 2,6-diaminopurine. Kornberg, A., DNA Replication, W. H. Freeman & Co., San Francisco, 1980, pp 75-′7′7; Gebeyehu, G., et al. Nucl. Acids Res. 1987, 15:4513). A “universal” base known in the art, e.g., inosine, can also be included. 5-Me-C substitutions can also be included. These have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278). Further suitable modified bases are described in U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175, 273; 5, 367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,596,091; 5,614,617; 5,750,692, and 5,681,941, each of which is herein incorporated by reference.

It is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the aforementioned modifications may be incorporated in a single oligonucleotide or even at a single nucleoside within an oligonucleotide.

In certain embodiments, both a sugar and an internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, for example, an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative United States patents that teach the preparation of PNA compounds comprise, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

In certain embodiments, the miRNA agent is linked (covalently or non-covalently) to one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include, without limitation, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Mancharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937), each of which is herein incorporated by reference in its entirety. See also U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552, 538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486, 603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762, 779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082, 830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5, 245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporated by reference in its entirety.

In one particular embodiment, the miRNA agent is linked to (covalently or non-covalently) to a nucleic acid aptamer. Aptamers are synthetic oligonucleotides or peptide molecules that bind to a specific target molecule. Aptamers appropriate for use with the miRNA agents provided herein are described in U.S. Provisional Patent Application No. 61/695,477 filed Aug. 31, 2012 and incorporated by reference herein in its entirety.

Accordingly, in a first aspect, the invention provides an adipocyte-specific miRNA modulator composition comprising i) a targeting moiety that selectively binds to a cellular surface marker on an adipose target cell in a human and ii) a thermogenic miRNA modulator moiety, wherein the targeting moiety facilitates uptake of the miRNA modulatory moiety by the target cell such that the miRNA is capable of targeting a thermogenic pathway and upregulating thermogenesis in the target cell.

In one embodiment, the composition comprises an aptamir comprising an aptamer as the targeting moiety.

In certain embodiments, the aptamers used with the miRNAs disclosed herein specifically bind to cell surface marker proteins on an adipose tissue mesenchymal stem cell (ATMSC), white adipose tissue (WAT) adipocytes and brown adipose tissue (BAT) adipocytes. Cell surface markers for ATMSCs include CD9, CD10, CD13, CD29, CD36, CD44, CD49d, CD54, CD55, CD59, CD73, CD90, CD91, CD105, CD137, CD146, CD166, and HLA-ABC. Cell surface markers for WAT adipocytes include Adiponectin, Caveolin-1, Caveolin-2, CD36 (FAT), CLH-22 (Clathrin Heavy Chain Chr 22), FABP4 (Adipocyte protein 2, aP2), SLC27A1 (FATP1), SLC27A2 (FATP2), GLUT4 (Glucose Transporter 4), Perilipin 2 or Resistin. Cell surface markers for all adipocytes include Neprilysin (CD10), FAT (CD36), Thy-1 (CD90), Low density lipoprotein receptor-related protein 1 (LRP1 or CD91), Caveolin-1, Caveolin-2, Fatty acid binding protein 4 (FABP4), Cell surface glycoprotein MUC18 (CD146), Activated leukocyte cell adhesion molecule (CD166) and Natriuretic peptide receptor A (NPR1). According to other embodiments, the aptamers for use with the miRNAs disclosed herein can also specifically bind to markers of adipose tissue including adiponectin, leptin, resistin, FGF 17, FGF 19, BMP7, PYY, MetAP2, RBP4, endostatin, and angiostatin.

In certain embodiments, the aptamers are selected by the Cell-SELEX technology, which uses whole living cells as the target, whereby aptamers that recognize specific molecules in their native conformation in their natural environment on the surface of intact cells are selected by repeated amplification and binding to living cells. In this cell-based selection, specific cell surface molecules or even unknown membrane receptors can be directly targeted within their native environment, allowing a straightforward enrichment of cell-specific aptamers.

In certain exemplary embodiments, the miRNA modulator is combined with an aptamer to create an “AptamiR” composition. There are many different ways to combine an aptamer and miRNA analog(s) to create an aptamir. They include, for example, aptamer-miRNA analog chimeras, aptamer-splice-switching oligonucleotide chimeras, and aptamer conjugated to nanoparticles or liposomes containing the miRNA analog(s). “Escort Aptamers” may be inserted at the surface of functional polymers, liposomes, and nanoparticles, each of which can carry many miRNA analogs. For instance, the size of thioaptamer-conjugated liposomes is about 120 nm. Nanoparticle approaches have several functional advantages, including, for example, cellular uptake, the ability to cross membranes, and triggered nanoparticle disassembly.

In one embodiment, an aptamiR compostion comprises an aptamer that is directly linked or fused to a miRNA modulator. Such aptamiRs are entirely chemically synthesized, which provides more control over the composition of the conjugate. For instance, the stoichiometry (ratio of miRNA analog per aptamer) and site of attachment can be precisely defined. The linkage portion of the conjugate presents a plurality (2 or more) of nucleophilic and/or electrophilic moieties that serve as the reactive attachment point for the aptamers and miRNA analogs. In addition, the aptamir may further comprise a linker between the aptamer and the miRNA analog. In some embodiments, the linker is a polyalkylene glycol, particularly a polyethylene glycol. In other embodiments, the linker is a liposome, exosome, dendrimer, or comb polymer. Other linkers can mediate the conjugation between the aptamer and the miRNA analog, including a biotinstreptavidin bridge, or a ribonucleic acid. Exemplary non-covalent linkers include linkers formed by base pairing a single stranded portion or overhang of the miRNA moiety and a complementary single-stranded portion or overhang of the aptamer moiety.

In another particular embodiment, an aptamer is combined with a miRNA analog in the form of a liposome-based aptamiR. Liposomes are spherical nanostructures made of a lipid bilayer that can be loaded with pharmaceuticals, such as miRNAs. Furthermore, the liposome surface can be loaded with different substances, such as polyethylene glycol (extending their systemic half life) or molecular recognition moieties like aptamers for specific binding to targeted cells. For example, aptamer-modified liposomes have been developed, with each liposome displaying approximately 250 aptamers tethered to its surface to facilitate target binding. In a preferred embodiment, liposomes are created to encapsulate miRNA analog(s) and display at their surface aptamers that specifically bind with high affinity and specificity to molecules (e.g. lipid transporters) highly expressed at the surface of adipocytes and ATMSCs. The fusion of the liposomes with the targeted cells causes the release of the miRNA analog(s) into the cell cytoplasm, which then alter a specific intra-cellular pathway. Alternatively, stable thioaptamers may be inserted at the surface of liposomes to guide delivery of the liposome miRNA analog(s) load to targeted ATMSCs and adipocytes.

In a further particular embodiment, an aptamer is combined with a miRNA analog in the form of a carrier-based aptamiR. Exemplary carriers include nanoparticles, lipsomes or exosomes. Such carrier-based aptamiR compositions have the capability of delivering a cargo of multiple miRNA modulators to the target cell in a single carrier. To accomplish targeting and accumulation, the carriers are formulated to present the targeting moiety on their external surface so they can react/bind with selected cell surface antigens or receptors on the adipose target cell. As an example, carriers may be created to encapsulate miRNA modulators while displaying at their surface aptamers that specifically bind with high affinity and specificity to molecules (e.g. lipid transporters) highly expressed at the surface of adipocytes and ATMSCs. The internalized exosomes release inside the cell cytoplasm their miRNA analog(s) load, which alters a specific intra-cellular pathway.

In one embodiment, the carrier is an exosome. Exosomes, which originate from late endosomes, are naturally occurring nanoparticles that are specifically loaded with proteins, mRNAs, or miRNAs, and are secreted endogenously by cells. Exosomes are released from host cells, are not cytotoxic, and can transfer information to specific cells based on their composition and the substance in/on the exosome. Because exosomes are particles of approximately 20-100 nm in diameter, the exosomes evade clearance by the mononuclear phagocyte system (which clears circulating particles >100 nm in size), and are very efficiently delivered to target tissues.

Moreover, synthetic exosomes may offer several advantages over other carriers. For example, they may deliver their cargo directly into the cytosol, while their inertness avoids attack and clearance in the extracellular environment. The structural constituents of exosomes may include small molecules responsible for processes like signal transduction, membrane transport, antigen presentation, targeting/adhesion, among many others.

The miRNA agents must be sufficiently complementary to the target mRNA, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired effect. “Complementary” refers to the capacity for pairing, through hydrogen bonding, between two sequences comprising naturally or non-naturally occurring bases or analogs thereof. For example, if a base at one position of a miRNA agent is capable of hydrogen bonding with a base at the corresponding position of a target nucleic acid sequence, then the bases are considered to be complementary to each other at that position. In certain embodiments, 100% complementarity is not required. In other embodiments, 100% complementarity is required.

miRNA agents for use in the methods disclosed herein can be designed using routine methods. While the specific sequences of certain exemplary target nucleic acid sequences and miRNA agents are set forth herein, one of skill in the art will recognize that these serve to illustrate and describe particular embodiments within the scope of the present invention. Additional target segments are readily identifiable by one having ordinary skill in the art in view of this disclosure. Target segments of 5, 6, 7, 8, 9, 10 or more nucleotides in length comprising a stretch of at least five (5) consecutive nucleotides within the seed sequence, or immediately adjacent thereto, are considered to be suitable for targeting a gene. In some embodiments, target segments can include sequences that comprise at least the 5 consecutive nucleotides from the 5 ‘-terminus of one of the seed sequence (the remaining nucleotides being a consecutive stretch of the same RNA beginning immediately upstream of the 5’-terminus of the seed sequence and continuing until the miRNA agent contains about 5 to about 30 nucleotides). In some embodiments, target segments are represented by RNA sequences that comprise at least the 5 consecutive nucleotides from the 3′-terminus of one of the seed sequence (the remaining nucleotides being a consecutive stretch of the same miRNA beginning immediately downstream of the 3′-terminus of the target segment and continuing until the miRNA agent contains about 5 to about 30 nucleotides). One having skill in the art armed with the sequences provided herein will be able, without undue experimentation, to identify further preferred regions to target using miRNA agents. Once one or more target regions, segments or sites have been identified, inhibitory nucleic acid compounds are chosen that are sufficiently complementary to the target, i.e., that hybridize sufficiently well and with sufficient specificity (i.e., do not substantially bind to other non-target nucleic acid sequences), to give the desired effect.

In certain embodiments, miRNA agents used to practice this invention are expressed from a recombinant vector. Suitable recombinant vectors include, without limitation, DNA plasmids, viral vectors or DNA minicircles. Generation of the vector construct can be accomplished using any suitable genetic engineering techniques well known in the art, including, without limitation, the standard techniques of PCR, oligonucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al. Molecular Cloning: A Laboratory Manual. (1989), Coffin et al. (Retroviruses. (1997) and “RNA Viruses: A Practical Approach” (Alan J. Cann, Ed., Oxford University Press, (2000)). As will be apparent to one of ordinary skill in the art, a variety of suitable vectors are available for transferring nucleic acids of the invention into cells. The selection of an appropriate vector to deliver nucleic acids and optimization of the conditions for insertion of the selected expression vector into the cell, are within the scope of one of ordinary skill in the art without the need for undue experimentation. Viral vectors comprise a nucleotide sequence having sequences for the production of recombinant virus in a packaging cell. Viral vectors expressing nucleic acids of the invention can be constructed based on viral backbones including, but not limited to, a retrovirus, lentivirus, adenovirus, adeno-associated virus, pox virus or alphavirus. The recombinant vectors can be delivered as described herein, and persist in target cells (e.g., stable transformants).

In certain embodiments, miRNA agents used to practice this invention are synthesized in vitro using chemical synthesis techniques, as described in, e.g., Adams (1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers (1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; U.S. Pat. No. 4,458,066, each of which is herein incorporated by reference in its entirety.

IV. Methods of Treatment

In one aspect, the invention provides a method of treating obesity in human subject. The method generally comprises administering to the human subject an effective amount of a miRNA agent that modulates activity of at least one thermogenic regulator, (e.g., a mitochondrial uncoupler, such as UCP1 and/or UCP2).

Such methods of treatment may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics. Thus, another aspect of the invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the target gene molecules of the present invention or target gene modulators according to that individual's drug response genotype. Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.

miRNA agents can be tested in an appropriate animal model e.g., an obesity model including ob/ob mice (Lindstrom P., ScientificWorld Journal, 7:666-685 (2007) and db/db mice (Sharma K et al., Am J Physiol Renal Physiol., 284 (6):F1138-1144 (2003)). For example, a miRNA agent (or expression vector or transgene encoding same) as described herein can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with said agent. Alternatively, a therapeutic agent can be used in an animal model to determine the mechanism of action of such an agent. For example, a miRNA agent can be used in an animal model to determine the efficacy, toxicity, or side effects of treatment with such an agent. Alternatively, an agent can be used in an animal model to determine the mechanism of action of such an agent.

The disclosure also provides a method of inducing pre-adipocytes to differentiate into white adipocytes and white adipocytes into brown adipocytes, comprising administering to a population of pre-adipocytes one or more miRNAs selected from hsa-let-7a agomir, hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-1 antagomir, hsa-miR-19 agomir, hsa-miR-19b agomir, hsa-miR-19b antagomir, hsa-miR-30b agomir and hsa-miR-30b antagomir. In certain embodiments, the induction of pre-adipocytes to differentiate into adipocytes is greater than the differentiation of pre-adipocytes to adipocytes than when pre-adipocytes are exposed to 100 mM rosiglitazone for two days followed by maintenance medium. In certain embodiments, the adipocytes are brown adipocytes. In other embodiments, the adipocytes are white adipocytes.

The disclosure also provides a method for increasing insulin sensitivity in a subject in need thereof comprising administering the subject one or more miRNAs selected from the group consisting of hsa-let-7a agomir, hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-1 antagomir, hsa-miR-19 agomir, hsa-miR-19b agomir, hsa-miR-19b antagomir, hsa-miR-30b agomir and hsa-miR-30b antagomir. In certain embodiments, the subject is a mammal.

The disclosure also provides a method of causing fat loss in a subject in need thereof comprising administering the subject one or more miRNAs selected from the group consisting of hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-19b agomir and hsa-miR-30b agomir. In certain embodiments, the subject is a mammal. In other embodiments, the mammal is a human.

A miRNA agent modified for enhancing uptake into cells (e.g., adipose cells) can be administered at a unit dose less than about 15 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, and less than 200 nmole of miRNA agent (e.g., about 4.4×10¹⁶ copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNA silencing agent per kg of bodyweight. The unit dose, for example, can be administered by injection (e.g., intravenous or intramuscular), an inhaled dose, or a topical application. Particularly preferred dosages are less than 2, 1, or 0.1 mg/kg of body weight. Delivery of a miRNA agent directly to an organ or tissue (e.g., directly to adipose tissue) can be at a dosage on the order of about 0.00001 mg to about 3 mg per organ/tissue, or preferably about 0.0001-0.001 mg per organ/tissue, about 0.03-3.0 mg per organ/tissue, about 0.1-3.0 mg per organ/tissue or about 0.3-3.0 mg per organ/tissue. The dosage can be an amount effective to treat or prevent obesity or to increase insulin sensitivity. In one embodiment, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time. In one embodiment, the effective dose is administered with other traditional therapeutic modalities.

In certain embodiment, a subject is administered an initial dose, and one or more maintenance doses of a miRNA agent. The maintenance dose or doses are generally lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 mg/kg to 1.4 mg/kg of body weight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. The maintenance doses are preferably administered no more than once every 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient. In preferred embodiments the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once every 5 or 8 days. Following treatment, the patient can be monitored for changes in condition, e.g., changes in percentage body fat. The dosage of the compound may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if a decrease in body fat is observed, or if undesired side-effects are observed.

The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stent (e.g., sub-cutaneous, intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable. In one embodiment, a pharmaceutical composition includes a plurality of miRNA agent species. In another embodiment, the miRNA agent species has sequences that are non-overlapping and non-adjacent to another species with respect to a naturally occurring target sequence. In another embodiment, the plurality of miRNA agent species is specific for different naturally occurring target genes. In another embodiment, the miRNA agent is allele specific. In another embodiment, the plurality of miRNA agent species target two or more SNP alleles (e.g., two, three, four, five, six, or more SNP alleles).

Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound of the invention is administered in maintenance doses, ranging from 0.01 mg per kg to 100 mg per kg of body weight (see U.S. Pat. No. 6,107,094).

The concentration or amount of miRNA agent administered will depend on the parameters determined for the agent and the method of administration, e.g. nasal, buccal, or pulmonary. For example, nasal formulations tend to require much lower concentrations of some ingredients in order to avoid irritation or burning of the nasal passages. It is sometimes desirable to dilute an oral formulation up to 10-100 times in order to provide a suitable nasal formulation.

Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a miRNA agent can include a single treatment or, preferably, can include a series of treatments. It will also be appreciated that the effective dosage of a miRNA agent for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein. For example, the subject can be monitored after administering a miRNA agent composition. Based on information from the monitoring, an additional amount of the miRNA agent composition can be administered.

Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In some embodiments, the animal models include transgenic animals that express a human gene, e.g., a gene that produces a target mRNA (e.g., a thermogenic regulator). The transgenic animal can be deficient for the corresponding endogenous mRNA. In another embodiment, the composition for testing includes a miRNA agent that is complementary, at least in an internal region, to a sequence that is conserved between a nucleic acid sequence in the animal model and the target nucleic acid sequence in a human.

Several studies have reported successful mammalian dosing using miRNA agents. For example, Esau C, et al., Cell Metabolism, 3(2): 87-98 (2006) reported dosing of normal mice with intraperitoneal doses of miR-122 antisense oligonucleotide ranging from 12.5 to 75 mg/kg twice weekly for 4 weeks. The mice appeared healthy and normal at the end of treatment, with no loss of body weight or reduced food intake. Plasma transaminase levels were in the normal range (AST ¾ 45, ALT ¾ 35) for all doses with the exception of the 75 mg/kg dose of miR-122 ASO, which showed a very mild increase in ALT and AST levels. They concluded that 50 mg/kg was an effective, nontoxic dose. Another study by Krutzfeldt J., et al., Nature, 438, 685-689 (2005), injected antagomirs to silence miR-122 in mice using a total dose of 80, 160 or 240 mg per kg body weight. The highest dose resulted in a complete loss of miR-122 signal. In yet another study, locked nucleic acids (“LNAs”) were successfully applied in primates to silence miR-122. Elmen J., et al., (2008) Nature 452, 896-899, report that efficient silencing of miR-122 was achieved in primates by three doses of 10 mg per kg LNA-antimiR, leading to a long-lasting and reversible decrease in total plasma cholesterol without any evidence for LNA-associated toxicities or histopathological changes in the study animals.

In certain embodiments, miRNA agents used to practice this invention are administered through expression from a recombinant vector. Suitable recombinant vectors include, without limitation, DNA plasmids, viral vectors or DNA minicircles. Generation of the vector construct can be accomplished using any suitable genetic engineering techniques well known in the art, including, without limitation, the standard techniques of PCR, oligonucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification, and DNA sequencing, for example as described in Sambrook et al. Molecular Cloning: A Laboratory Manual. (1989)), Coffin et al. (Retroviruses. (1997)) and “RNA Viruses: A Practical Approach” (Alan J. Cann, Ed., Oxford University Press, (2000)). As will be apparent to one of ordinary skill in the art, a variety of suitable vectors are available for transferring nucleic acids of the invention into cells. The selection of an appropriate vector to deliver nucleic acids and optimization of the conditions for insertion of the selected expression vector into the cell, are within the scope of one of ordinary skill in the art without the need for undue experimentation. Viral vectors comprise a nucleotide sequence having sequences for the production of recombinant virus in a packaging cell. Viral vectors expressing nucleic acids of the invention can be constructed based on viral backbones including, but not limited to, a retrovirus, lentivirus, adenovirus, adeno-associated virus, pox virus or alphavirus. The recombinant vectors can be delivered as described herein, and persist in target cells (e.g., stable transformants).

miRNA agents may be directly introduced into a cell (e.g., an adipocyte); or introduced extracellularly into a cavity, interstitial space, into the circulation of an organism, introduced orally, or may be introduced by bathing a cell or organism in a solution containing the nucleic acid. Vascular or extravascular circulation, the blood or lymph system, and the cerebrospinal fluid are sites where the nucleic acid may be introduced.

The miRNA agents of the invention can be introduced using nucleic acid delivery methods known in art including injection of a solution containing the nucleic acid, bombardment by particles covered by the nucleic acid, soaking the cell or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the nucleic acid. Other methods known in the art for introducing nucleic acids to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, and the like. The miRNA agents may be introduced along with other components e.g., compounds that enhance miRNA agent uptake by a cell.

In certain embodiments, the methods described herein include co-administration of miRNA agents with other drugs or pharmaceuticals, e.g., compositions for modulating thermogenesis, compositions for treating diabetes, compositions for treating obesity. Compositions for modulating thermogenesis include beta-3 adrenergic agonists, thyroid hormones, PPARG agonists, leptin, adiponectin, and orexin.

V. Screening Methods

In another aspect, the invention provides a method of screening for a miRNA agent that modulates thermogenesis, decreases obesity, or improves insulin sensitivity. The method generally comprises the steps of: providing an indicator cell; contacting the indicator cell with a test miRNA agent; and determining the expression level and/or cellular activity of at least one thermogenic regulator in the indicator cell in the presence and absence of the miRNA agent, wherein a change in the activity of the thermogenic regulator in the presence of the test miRNA agent identifies the test miRNA agent as a miRNA agent that modulates thermogenesis, decreases obesity, or improves insulin sensitivity. In certain embodiments, the method involves determining an increase the expression level and/or activity of the thermogenic regulator (e.g., UCP1, UCP2). The indicator cell can be a mammalian cell. In certain embodiments, the mammalian cell is a human cell, which comprises at least a portion of a human genome.

Any thermogenic regulator can be assayed in the methods disclosed herein. Exemplary thermogenic regulators are set forth in Table 1. In a preferred embodiment, the thermogenic regulator is a mitochondrial uncoupling protein e.g., UCP1 and/or UCP2.

Any cell in which the activity of a thermogenic regulator can be measured is suitable for use in the methods disclosed herein. Exemplary cells include pre-adipocytes, adipocytes, adipose tissue derived mesenchymal stem cells, hepatocytes, myocytes, or precursors thereof.

Any activity of a thermogenic regulator can be assayed, including, without limitation, mRNA expression level, protein expression level or mitochondrial uncoupling activity of the thermogenic regulator. Methods for determining such activities are well known in the art.

Any miRNA agent can be screened, including, without limitation, miRNA, agomirs, antagomirs, aptamirs, miR-masks, miRNA sponges, siRNA (single- or double-stranded), shRNA, antisense oligonucleotides, ribozymes, or other oligonucleotide mimetics which hybridize to at least a portion of a target nucleic acid and modulate its function.

VI. Pharmaceutical Compositions

In one aspect, the methods disclosed herein can include the administration of pharmaceutical compositions and formulations comprising miRNA agents capable of modulating the activity of at least one thermogenic modulator.

In certain embodiments, the compositions are formulated with a pharmaceutically acceptable carrier. The pharmaceutical compositions and formulations can be administered parenterally, topically, by direct administration into the gastrointestinal tract (e.g., orally or rectally), or by local administration, such as by aerosol or transdermally. The pharmaceutical compositions can be formulated in any way and can be administered in a variety of unit dosage forms depending upon the condition or disease and the degree of illness, the general medical condition of each patient, the resulting preferred method of administration and the like. Details on techniques for formulation and administration of pharmaceuticals are well described in the scientific and patent literature, see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005.

The miRNA agents can be administered alone or as a component of a pharmaceutical formulation (composition). The compounds may be formulated for administration, in any convenient way for use in human or veterinary medicine. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Formulations of the compositions of the invention include those suitable for intradermal, inhalation, oral/nasal, topical, parenteral, rectal, and/or intravaginal administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of active ingredient (e.g., nucleic acid sequences of this invention) which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration, e.g., intradermal or inhalation. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect, e.g., an antigen specific T cell or humoral response.

Pharmaceutical formulations of the invention can be prepared according to any method known to the art for the manufacture of pharmaceuticals. Such drugs can contain sweetening agents, flavoring agents, coloring agents and preserving agents. A formulation can be admixtured with nontoxic pharmaceutically acceptable excipients which are suitable for manufacture. Formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, on patches, in implants, etc.

Pharmaceutical formulations for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in appropriate and suitable dosages. Such carriers enable the pharmaceuticals to be formulated in unit dosage forms as tablets, pills, powder, dragées, capsules, liquids, lozenges, gels, syrups, slurries, suspensions, etc., suitable for ingestion by the patient. Pharmaceutical preparations for oral use can be formulated as a solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable additional compounds, if desired, to obtain tablets or dragée cores. Suitable solid excipients are carbohydrate or protein fillers include, e.g., sugars, including lactose, sucrose, mannitol, or sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxy-methylcellulose; and gums including arabic and tragacanth; and proteins, e.g., gelatin and collagen. Disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. Push-fit capsules can contain active agents mixed with a filler or binders such as lactose or starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active agents can be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycol with or without stabilizers.

Aqueous suspensions can contain an active agent (e.g., nucleic acid sequences of the invention) in admixture with excipients suitable for the manufacture of aqueous suspensions, e.g., for aqueous intradermal injections. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan mono-oleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin. Formulations can be adjusted for osmolarity.

In certain embodiments, oil-based pharmaceuticals are used for administration of the miRNA agents. Oil-based suspensions can be formulated by suspending an active agent in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin; or a mixture of these. See e.g., U.S. Pat. No. 5,716,928 describing using essential oils or essential oil components for increasing bioavailability and reducing inter- and intra-individual variability of orally administered hydrophobic pharmaceutical compounds (see also U.S. Pat. No. 5,858,401). The oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents can be added to provide a palatable oral preparation, such as glycerol, sorbitol or sucrose. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid. As an example of an injectable oil vehicle, see Minto (1997) J. Pharmacol. Exp. Ther. 281:93-102.

In certain embodiments, the pharmaceutical compositions and formulations are in the form of oil-in-water emulsions. The oily phase can be a vegetable oil or a mineral oil, described above, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsion can also contain sweetening agents and flavoring agents, as in the formulation of syrups and elixirs. Such formulations can also contain a demulcent, a preservative, or a coloring agent. In alternative embodiments, these injectable oil-in-water emulsions of the invention comprise a paraffin oil, a sorbitan monooleate, an ethoxylated sorbitan monooleate and/or an ethoxylated sorbitan trioleate.

In certain embodiments, the pharmaceutical compositions and formulations are administered by in intranasal, intraocular and intravaginal routes including suppositories, insufflation, powders and aerosol formulations (for examples of steroid inhalants, see e.g., Rohatagi (1995) J. Clin. Pharmacol. 35: 1187-1193; Tjwa (1995) Ann. Allergy Asthma Immunol. 75: 107-111). Suppositories formulations can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at body temperatures and will therefore melt in the body to release the drug. Such materials are cocoa butter and polyethylene glycols.

In certain embodiments, the pharmaceutical compositions and formulations are delivered transdermally, by a topical route, formulated as applicator sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, and aerosols.

In certain embodiments, the pharmaceutical compositions and formulations are delivered as microspheres for slow release in the body. For example, microspheres can be administered via intradermal injection of drug which slowly release subcutaneously; see Rao (1995) J. Biomater Sci. Polym. Ed. 7:623-645; as biodegradable and injectable gel formulations, see, e.g., Gao (1995) Pharm. Res. 12:857-863 (1995); or, as microspheres for oral administration, see, e.g., Eyles (1997) J. Pharm. Pharmacol. 49:669-674.

In certain embodiments, the pharmaceutical compositions and formulations are parenterally administered, such as by intravenous (IV) administration or administration into a body cavity or lumen of an organ. These formulations can comprise a solution of active agent dissolved in a pharmaceutically acceptable carrier. Acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can be employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables. These solutions are sterile and generally free of undesirable matter. These formulations may be sterilized by conventional, well known sterilization techniques. The formulations may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight, and the like, in accordance with the particular mode of administration selected and the patient's needs. For IV administration, the formulation can be a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated using those suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a suspension in a nontoxic parenterally-acceptable diluent or solvent, such as a solution of 1,3-butanediol. The administration can be by bolus or continuous infusion (e.g., substantially uninterrupted introduction into a blood vessel for a specified period of time). In certain embodiments, the pharmaceutical compounds and formulations are lyophilized.

Stable lyophilized formulations comprising an inhibitory nucleic acid can be made by lyophilizing a solution comprising a pharmaceutical of the invention and a bulking agent, e.g., mannitol, trehalose, raffinose, and sucrose or mixtures thereof. A process for preparing a stable lyophilized formulation can include lyophilizing a solution about 2.5 mg/mL nucleic acid, about 15 mg/mL sucrose, about 19 mg/mL NaCl, and a sodium citrate buffer having a pH greater than 5.5 but less than 6.5. See, e.g., U.S. 20040028670.

In certain embodiments, the pharmaceutical compositions and formulations are delivered by the use of liposomes. By using liposomes, particularly where the liposome surface carries ligands specific for target cells, or are otherwise preferentially directed to a specific organ, one can focus the delivery of the active agent into target cells in vivo. See, e.g., U.S. Pat. Nos. 6,063,400; 6,007,839; Al-Muhammed (1996) J. Microencapsul. 13:293-306; Chonn (1995) Curr. Opin. Biotechnol. 6:698-708; Ostro (1989) Am. J. Hosp. Pharm. 46: 1576-1587.

The formulations of the invention can be administered for prophylactic and/or therapeutic treatments. In certain embodiments, for therapeutic applications, compositions are administered to a subject who is need of reduced triglyceride levels, or who is at risk of or has a disorder described herein, in an amount sufficient to cure, alleviate or partially arrest the clinical manifestations of the disorder or its complications; this can be called a therapeutically effective amount. For example, in certain embodiments, pharmaceutical compositions of the invention are administered in an amount sufficient to treat obesity in a subject.

The amount of pharmaceutical composition adequate to accomplish this is a therapeutically effective dose. The dosage schedule and amounts effective for this use, i.e., the dosing regimen, will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age and the like. In calculating the dosage regimen for a patient, the mode of administration also is taken into consideration.

The dosage regimen also takes into consideration pharmacokinetics parameters well known in the art, i.e., the active agents' rate of absorption, bioavailability, metabolism, clearance, and the like (see, e.g., Hidalgo-Aragones (1996) J. Steroid Biochem. Mol. Biol. 58:611-617; Groning (1996) Pharmazie 51:337-341; Fotherby (1996) Contraception 54:59-69; Johnson (1995) J. Pharm. Sci. 84: 1144-1146; Rohatagi (1995) Pharmazie 50:610-613; Brophy (1983) Eur. J. Clin. Pharmacol. 24: 103-108; Remington: The Science and Practice of Pharmacy, 21st ed., 2005). The state of the art allows the clinician to determine the dosage regimen for each individual patient, active agent and disease or condition treated. Guidelines provided for similar compositions used as pharmaceuticals can be used as guidance to determine the dosage regiment, i.e., dose schedule and dosage levels, administered practicing the methods of the invention are correct and appropriate. Single or multiple administrations of formulations can be given depending on for example: the dosage and frequency as required and tolerated by the patient, the degree and amount of cholesterol homeostasis generated after each administration, and the like. The formulations should provide a sufficient quantity of active agent to effectively treat, prevent or ameliorate conditions, diseases or symptoms, e.g., treat obesity.

In certain embodiments, pharmaceutical formulations for oral administration are in a daily amount of between about 1 to 100 or more mg per kilogram of body weight per day. Lower dosages can be used, in contrast to administration orally, into the blood stream, into a body cavity or into a lumen of an organ. Substantially higher dosages can be used in topical or oral administration or administering by powders, spray or inhalation. Actual methods for preparing parenterally or non-parenterally administrable formulations will be known or apparent to those skilled in the art and are described in more detail in such publications as Remington: The Science and Practice of Pharmacy, 21st ed., 2005.

VII. Exemplification

The present invention is further illustrated by the following examples which should not be construed as further limiting. The contents of Sequence Listing, figures and all references, patents and published patent applications cited throughout this application are expressly incorporated herein by reference.

Furthermore, in accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein “Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization [B. D. Hames & S. J. Higgins eds. (1985)]; Transcription And Translation [B. D. Hames & S. J. Higgins, eds. (1984)]; Animal Cell Culture [R. I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

Example 1 In-Silico Analysis of Thermogenic Regulators

Eighty three proteins that are involved in regulation of thermogenesis were selected based upon a critical assessment and review of the available scientific information and our own experimental data. These proteins were categorized as activators or repressors of thermogenesis based upon their functions. These thermogenic regulator proteins are set forth in Table 1.

TABLE 1 Thermogenic regulator proteins. Name Entrez Gene ID Ensembl Gene ID Activators 1 ALDH1A1 216 ENSG00000165092 2 ANP (NPPA) 4878 ENSG00000175206 3 AZGP1 563 ENSG00000160862 4 BMP7 655 ENSG00000101144 5 BMP8b 656 ENSG00000116985 6 CEBPA 1050 ENSG00000245848 7 CEBPB 1051 ENSG00000172216 8 CEBPD 1052 ENSG00000221869 9 CIDEA 1149 ENSG00000176194 10 COX7A1 1346 ENSG00000161281 11 CRAT 1384 ENSG00000095321 12 CREB1 1385 ENSG00000118260 13 CREBBP 1387 ENSG00000005339 14 CTBP1 1487 ENSG00000159692 15 CTBP2 1488 ENSG00000175029 16 DIO2 1734 ENSG00000211448 17 ELOVL3 83401 ENSG00000119915 18 FGF16 8823 ENSG00000196468 19 FGF19 9965 ENSG00000162344 20 FGF21 26291 ENSG00000105550 21 FNDC5 252995 ENSG00000160097 22 FOXC2 2303 ENSG00000176692 23 GDF3 9573 ENSG00000184344 24 HCRT (OREXIN) 3060 ENSG00000161610 25 HOXC8 3224 ENSG00000037965 26 INSR 3643 ENSG00000171105 27 IRS1 3667 ENSG00000169047 28 KDM3A (JMJD1A) 55818 ENSG00000115548 29 KLF5 688 ENSG00000102554 30 KLF11 8462 ENSG00000172059 31 KLF15 28999 ENSG00000163884 32 LRP6 4040 ENSG00000070018 33 MAPK14 1432 ENSG00000112062 34 MED13 9969 ENSG00000108510 35 NCOA1 8648 ENSG00000084676 36 NCOA2 10499 ENSG00000140396 37 NCOA3 8202 ENSG00000124151 38 NR4A3 8013 ENSG00000119508 39 NRF1 4899 ENSG00000106459 40 PLAC8 51316 ENSG00000145287 41 PPARA 5465 ENSG00000186951 42 PPARD 5467 ENSG00000112033 43 PPARG 5468 ENSG00000132170 44 PPARGC1A 10891 ENSG00000109819 45 PPARGC1B 133522 ENSG00000155846 46 PRDM16 63976 ENSG00000142611 47 PRDX3 10935 ENSG00000165672 48 PRKAA1 (AMPKA1) 5562 ENSG00000132356 49 PRKAA2 (AMPKA2) 5563 ENSG00000162409 50 PRKACA 5566 ENSG00000072062 51 PRKACB 5567 ENSG00000142875 52 PRKAR1A 5573 ENSG00000108946 53 SIRT1 23411 ENSG00000096717 54 SIRT3 23410 ENSG00000142082 55 SLC27A2 (FATP2) 11001 ENSG00000140284 56 SREBF1 6720 ENSG00000072310 58 SREBF2 6721 ENSG00000198911 58 STAT5A 6776 ENSG00000126561 59 TRPM8 79054 ENSG00000144481 60 UCP1 (SLC25A7) 7350 ENSG00000109424 61 UCP2 (SLC25A8) 7351 ENSG00000175567 62 UCP3 (SLC25A9) 7352 ENSG00000175564 Repressors 1 ATG7 10533 ENSG00000197548 2 BMP2 650 ENSG00000125845 3 BMP4 652 ENSG00000125378 4 CIDEC 63924 ENSG00000187288 5 CTNNB1 1499 ENSG00000168036 6 DLK1 (Pref-1) 8788 ENSG00000185559 7 E2F4 (p107) 1874 ENSG00000205250 8 EIF4EBP1 1978 ENSG00000187840 9 ESRRA (NR3B1) 2101 ENSG00000173153 10 IKBKE 9641 ENSG00000143466 11 NR1H3 (LXRA) 10062 ENSG00000025434 12 NRIP1 (RIP140) 8204 ENSG00000180530 13 RB1 (pRb) 5925 ENSG00000139687 14 NR0B2 (SHP) 8431 ENSG00000131910 15 RPS6KB1 6198 ENSG00000108443 16 RUNX1T1 862 ENSG00000079102 17 RUNX2 860 ENSG00000124813 18 TNFRSF1A 7132 ENSG00000067182 19 TWIST1 7291 ENSG00000122691 20 WNT5A 7474 ENSG00000114251 21 WNT10B 7480 ENSG00000169884

The STRING 9.0 database of known and predicted protein interactions (string-db.org/) was used to test these 83 candidate molecules. The interactions include direct (physical) and indirect (functional) associations; they are derived from four sources: genomic context; high-throughput experiments; co-expression; and previous knowledge. STRING quantitatively integrates interaction data from these sources for a large number of organisms, and transfers information between these organisms where applicable. The database currently covers 5,214,234 proteins from 1,133 organisms. As an example, the relationships between the 83 thermogenic regulator molecules were centered around UCP1, and molecules having direct and indirect connections with UCP1 could be distinguished using the highest confidence score of 0.90. This relationship is set forth in schematic form in FIG. 1A. From this analysis, it was discovered that nine molecules (CEBPB, CIDEA, KDM3A, NRIP1, PRDM16, PPARG, PPARGC1A, PPKAA2, and UCP2) are directly linked to UCP1, whereas many more molecules are connected to UCP1 on a second or higher degree order.

When the degree of confidence was set to high with a score of 0.70, eight additional proteins were found to be directly linked to UCP1 (AZGP1, DIO2, KLF11, KLF15, NR1H3, PPARA, PPARD, and PPARGC1B), FIG. 1B.

Similarly, the interactions among these 83 thermogenic regulator molecules were independently assessed using other software programs. The interactions predicted by the Ingenuity Pathway Analysis (IPA) Software program (www.ingenuity.com) are shown in FIG. 2A (UCP1 in yellow, activators in green and repressors in purple). The interactions predicted by the Reactome Functional Interaction (Reactome IF) Software program (http://wiki.reactome.org) are shown in FIG. 2B (UCP1 in yellow, activators in green and repressors in purple). The IPA and Reactome IF networks differ from the one set forth in FIG. 1, obtained with the STRING program. It is not surprising that the results of these algorithms are different because they rely on different predefined parameters, sources of information and selection criteria.

Example 2 In-Silico Selection of Relevant miRNA Targets

To select thermogenic regulators suitable as targets for miRNA agents, several internet-based resources were employed to match miRNAs and their targets (the “micronome”). Exemplary tools are set forth in Table 2A.

TABLE 2A Exemplary bioinformatics tools used to select miRNAs and their targets. Field & Name Function Web Address Integrated Data Mining (8) BioCarta Catalogs and summarizes important biocarta.com resources providing information for over 120,000 genes from multiple species. Find both classical pathways as well as current suggestions for new pathways Database for Integrated biological david.abcc.ncifcrf.gov/home.jsp Annotation, knowledgebase and analytic tools Visualization and aimed at systematically extracting Integrated biological meaning from large Discovery gene/protein lists (DAVID) GeneOntology Standardizing the representation of geneontology.org/ gene and gene product attributes across species and databases Gene Set Computational method that broadinstitute.org/gsea/index.jsp Enrichment determines whether an a priori Analysis (GSEA) defined set of genes shows statistically significant, concordant differences between two biological states (e.g. phenotypes). KEGG Kyoto Encyclopedia of Genes and genome.jp/kegg/ Genomes PubGene Connecting up-to-date information pubgene.org/ on genes and related terms Reactome An open-source, open access, reactome.org/ReactomeGWT/entrypoint.html manually curated and peer- reviewed pathway database. STRING Database of known and predicted string-db.org/ protein interactions; direct (physical) and indirect (functional) associations miRNA Mining & Mapping (8) deepBase Platform for annotating and deepbase.sysu.edu.cn/ discovering small and long ncRNAs (microRNAs, siRNAs, piRNAs . . . ) Human Contains miRNA names, disease 202.38.126.151/hmdd/mirna/md/ microRNA names, dysfunction evidences, and disease database the literature PubMed ID (HMDD) miRBase V19 Searchable database of published mirbase.org/ miRNA sequences and annotation miRGen 2.0 Database of microRNA genomic diana.cslab.ece.ntua.gr/mirgen/ information and regulation miRNAMap Experimentally verified microRNAs mirnamap.mbc.nctu.edu.tw/ Shows tissue expression profile miRSel Automated extraction of services.bio.ifi.lmu.de/mirsel/ associations between microRNAs and genes from the biomedical literature miRStart Database of human microRNA mirstart.mbc.nctu.edu.tw/home.php TSSs (transcription start sites) miR2Disease A manually curated database mir2disease.org providing a comprehensive resource of miRNA deregulation in various human diseases miRNA Targets & Expression (21) DIANA-microT Algorithm based on several diana.cslab.ece.ntua.gr/microT/ 3.0 parameters calculated individually for each microRNA and it combines conserved and non-conserved microRNA recognition elements into a final prediction score. DIANA- Algorithm that can identify diana.cslab.ece.ntua.gr/hexamers/ mirExTra microRNA effects to the Expression levels of protein-coding transcripts, based on the frequency of six nucleotide long motifs in the 3′UTR sequences of genes. GSEA Molecular Gene sets that contain genes sharing broadinstitute.org/gsea/index.jsp Signatures a 3′-UTR microRNA binding motif Database v3.0 (n = 221) MicroCosm Computationally predicted targets ebi.ac.uk/enright-srv/microcosm/cgi-bin/targets/v5/download.pl Targets for microRNAs across many species. The miRNA sequences are obtained from the miRBase Sequence database and most genomic sequence from EnsEMBL MicroInspector A scanning software for detection bioinfo.uni-plovdiv.bg/microinspector/ of miRNA binding sites using hybridization temperature and free energy cut-off value microRNA.org Predicted microRNA targets & microrna.org/microrna/home.do (ex. miRanda) target downregulation scores. Experimentally observed expression patterns. miRDB Online database for miRNA target mirdb.org/miRDB/ prediction and functional annotations in animals by a new bioinformatics tool analyzing thousands of genes impacted by miRNAs with an SVM learning machine. miRTarBase Has accumulated more than three mirtarbase.mbc.nctu.edu.tw/index.html thousand miRNA-target interactions (MTIs), which are collected by manually surveying pertinent literature miRTar.Human An integrated web server for mirtar.mbc.nctu.edu.tw/human/download.php identifying miRNA-target interactions. Identifies the biological functions and regulatory relationships between a group of known/putative miRNAs and protein coding genes. It also provides perspective of information on the miRNA targets on alternatively spliced transcripts in human miRvestigator Takes as input a list of co-expressed mirvestigator.systemsbiology.net/ genes and will return the most likely miRNA regulating these genes. It does this by searching for an over- represented sequence motif in the 3′UTRs of the genes using Weeder and then comparing this to the miRNA seed sequences in miRBase using our custom built miRvestigator hidden Markov model (HMM) mirZ A server that provides statistical mirz.unibas.ch/ElMMo2/ analysis and data mining tools operating on up-to-date databases of sequencing-based miRNA expression profiles and of predicted miRNA target sites MultiMiTar A Support Vector Machine (SVM) isical.ac.in/~bioinfo_miu/multimitar.htm based classifier integrated with a multiobjective metaheuristic based feature selection technique. PhenomiR Provides information about mips.helmholtz-muenchen.de/phenomir/index.gsp differentially regulated miRNA expression in diseases and other biological processes. The content of PhenomiR is completely generated by manual curation of experienced annotators. Data was extracted from more than 365 scientific articles and resulted in more than 632 database entries as of 02 2011 PicTar Algorithm for the identification of pictar.mdc-berlin.de/ microRNA targets. This searchable website provides details (3′ UTR alignments with predicted sites, links to various public databases, etc.) PITA Incorporates the role of target-site genie.weizmann.ac.il/pubs/mir07/mir07_data.html accessibility, as determined by base- pairing interactions within the mRNA, in microRNA target recognition. RepTar Database of miRNA target bioinformatics.ekmd.huji.ac.il/reptar/ predictions, based on an algorithm that is independent of evolutionary conservation considerations and is not limited to seed pairing sites. RNAhybrid A tool for finding the minimum free bibiserv.techfak.uni-bielefeld.de/rnahybrid/ energy hybridisation of a long and a short RNA. The hybridisation is performed in a kind of domain mode, ie. the short sequence is hybridised to the best fitting part of the long one. RNA22 First finds putative microRNA cbcsrv.watson.ibm.com/rna22.html binding sites in the sequence of interest, then identifies the targeted microRNA (IBM). Sylamer A system for finding significantly ebi.ac.uk/enright/sylamer/ over or under-represented words in sequences according to a sorted gene list. It is used to find significant enrichment or depletion of microRNA or siRNA seed sequences from microarray expression data. TarBase 6.0 Database of experimentally diana.cslab.ece.ntua.gr/DianaToolsNew/index.php?r=tarbase/index supported microRNA targets. TargetScanHuman Predicts biological targets of targetscan.org/ 6.2 miRNAs by searching for the presence of conserved 8mer and 7mer sites that match the seed region of each miRNA, using 6 features: site-type contribution, 3′ pairing contribution, local AU contribution, position contribution, TA (target site abundance) contribution, SPS (seed-pairing stability) contribution. Integrated miRNA Targets & Expression Tools (13) GOmir Integrates the predicted target genes bioacademy.gr/bioinformatics/projects/GOmir/ from TargetScan, miRanda, RNAhybrid and PicTar computational tools and also providing a full gene description and functional analysis for each target gene. MAMI Compiles predictions from five mami.med.harvard.edu/ (MetaMiR:Target different miRNA target prediction Inference) algorithms (TargetScanS, miRanda, microT, miRtarget, and picTar). mimiRNA Allows the visualization of miRNA mimirna.centenary.org.au/mep/formulaire.html expression levels in 188 different tissue or cell types, provides a robust statistical method for discovering functional interactions between miRNAs and mRNA genes. Uses a novel sample classification algorithm, ExParser, that allows mimiRNA to automatically classify imported experiments with minimal curation MMIA Integrates the predicted target genes 147.46.15.115/MMIA/index.html (microRNA and from TargetScan, PicTar, PITA mRNA Integrated Analysis) mirDIP Integrates twelve microRNA ophid.utoronto.ca/mirDIP/ prediction datasets from six microRNA prediction databases, allowing users to customize their microRNA target searches. Combining microRNA predictions allows users to obtain more robust target predictions, giving you more confidence in your microRNA targets. miRGator V3.0 Integrated database of miRNA- mirgator.kobic.re.kr associated gene expression, target prediction, disease association and genomic annotation, using mirBridge, miRanda, PITA and TargetScan. Now includes 73 deep sequencing datasets on human samples from GEO, SRA, and TCGA archives miRecords Integrates the predictions form mirecords.biolead.org/ DIANA-microT, MicroInspector, miRanda, mirTarget2, miTarget, NBmiRTar, PicTar, PITA, rna22, RNAhybrid, TargetScan/TargetScanS MIRNA- Automatically extracts miRNAs ikp-stuttgart.de/content/languagel/html/10415.asp DISTILLER predicted to interact with a given set of target genes from several selectable public databases. MiRonTop Online java web tool that integrates microarray.fr:8080/miRonTop/index DNA microarrays or high- throughput sequencing data to identify the potential implication of miRNAs on a specific biological system. It allows a rapid characterization of the most pertinent mRNA targets according to several existing miRNA target prediction approaches (Mirbase, miRanda, exact seed, TargetScan or PicTar) miRror Integrates predictions from a dozen proto.cs.huji.ac.il/mirror of miRNA resources that are based on complementary algorithms into a unified statistical framework. miRSystem Database which integrates 7 mirsystem.cgm.ntu.edu.tw/ miRNA target gene prediction programs: DIANA, miRanda, miRBridge, PicTar, PITA, rna22, and TargetScan. miRWalk Comprehensive database that ma.uni-heidelberg.de/apps/zmf/mirwalk/index.html provides information on miRNA on their predicted as well as validated binding sites on their target genes. StarBase Public platform for decoding starbase.sysu.edu.cn/index.php microRNA-target and protein-RNA interaction maps from CLIP-Seq (HITS-CLIP, PAR-CLIP) and degradome sequencing (Degradome-Seq, PARE) data. miRNA Secondary Structure (5) OligoWalk An online server calculating rna.urmc.rochester.edu/cgi-bin/server_exe/oligowalk/oligowalk_form.cgi thermodynamic features of sense- antisense hybridization. It predicts the free energy changes of oligonucleotides binding to a target RNA. It can be used to design efficient siRNA targeting a given mRNA sequence. PicTar RNA The BiBiServ Tool section offers www.pictar.org/ Studio bioinformatics tools for a large variety of tasks, including RNA studio RNA2D Suite of programs for discovering protein3d.ncifcrf.gov/shuyun/rna2d.html structural features in RNAs. Vienna RNA RNA Secondary Structure tbi.univie.ac.at/ivo/RNA/ Package Prediction and Comparison. Whitehead siRNA Helps select siRNAs to knock down jura.wi.mit.edu/bioc/siRNAext/ algorithm your gene of interest Network Searches & Analyses (8) ARIADNE Pathway analysis software helping ariadnegenomics.com/products/pathway-studio/ Pathway Studio to: Interpret gene expression and other high throughput data Build, expand and analyze pathways Find relationships among genes, proteins, cell processes and diseases Draw publication-quality diagrams Cytoscape Open source bioinformatics cytoscape.org/ software platform for visualizing molecular interaction networks and biological pathways and integrating these networks with annotations, gene expression profiles and other state data. Database for Integrated biological david.abcc.ncifcrf.gov/home.jsp Annotation, knowledgebase and analytic tools Visualization and aimed at systematically extracting Integrated biological meaning from large Discovery gene/protein lists (DAVID) Genego MetaCore An integrated knowledge database genego.com/metacore.php and software suite for pathway analysis of experimental data and gene lists based on a proprietary manually curated database of human protein-protein, protein-DNA and protein compound interactions, metabolic and signaling pathways. Ingenuity Systems To understand biology at multiple ingenuity.com/products/IPA/microRNA.html IPA levels by integrating data from a (Ingenuity variety of experimental platforms Pathway and providing insight into the Analysis) molecular and chemical interactions, cellular phenotypes, and disease processes. MATISSE A program for detection of acgt.cs.tau.ac.il/matisse/ (Module Analysis functional modules using interaction via Topology of networks and expression data Interactions and Similarity SEts) MIR@NT@N a framework integrating mironton.uni.lu transcription factors, microRNAs and their targets to identify sub- network motifs in a meta-regulation network model NAViGaTOR Network Analysis, Visualization, & ophid.utoronto.ca/navigator/index.html Graphing TORonto is a software package for visualizing and analyzing protein-protein interaction networks. Molecular Visualization (4) Foldit Multiplayer online game that enlists fold.it/portal/info/science players worldwide to solve difficult protein-structure prediction problems. PyMOL A user-sponsored molecular .pymol.org/ visualization system on an open- source foundation. Qlucore Omics To examine and analyze data from qlucore.com/ProdOverviewmiRNA.aspx Explorer miRNA experiments. WebMol Displays and analyzes structural cmpharm.ucsf.edu/cgi-bin/webmol.pl information contained in the Brookhaven Protein Data Bank (PDB). It can be run as an applet or as a stand-alone application. Information Integration (1) TIBCO Spotfire Comprehensive software platform that allows customers to analyze data, using predictive and complex statistics in the analysis

Specifically, these tools were used to perform: 1) Integrated Data Mining (8 tools); 2) miRNA Mining and Mapping (6 tools); 3) miRNA Target Targets and Expression (21 tools); 4) Integrated miRNA Targets and Expression (13 tools); 5) miRNA Secondary Structure Prediction and Comparison (5 tools); 6) Network Searches and Analyses (8 tools); 7) Molecular Visualization (4 tools); and 8) Information Integration and Exploitation (1 tool).

A single gene target can be controlled by several miRNAs whereas a single miRNA can control several gene targets. Sophisticated bioinformatics resources have been developed to select the most relevant miRNAs to target diseases (Gallagher I J, et al. Genome medicine. 2010; Fujiki K, et al. BMC Biol. 2009; Okada Y, et al., J Androl. 2010; Hao T, et al., Mol Biosyst. 2012; Hao T, et al., Mol Biosyst. 2012). However, the results of these algorithms are acutely dependent on predefined parameters and the degree of convergence between these algorithms is rather limited. Therefore, there is a need to develop better performing bioinformatics tools with improved sensitivity, specificity and selectivity for the identification of miRNA/target relationships.

The interactions between miRNAs and their targets go beyond the original description of miRNAs as post-transcriptional regulators whose seed region of the driver strand (5′ bases 2-7) bind to complementary sequences in the 3′ UTR region of target mRNAs, usually resulting in translational repression or target degradation and gene silencing. The interactions can also involve various regions of the driver or passenger strands of the miRNAs as well as the 5′UTR, promoter, and coding regions of the mRNAs.

Upon analysis of the available data, it was decided to favor pathway-specific miRNAs which target multiple genes within one discrete signaling pathway, rather than universal miRNAs which are involved in many signaling pathways, functions or processes. Using 34 publicly available Internet tools predicting miRNA targets, specific huma miRNAs were searched for that could potentially modulate several targets among the 83 thermogenic regulator molecules (which include 36 Transcription Factors) selected in Example 1.

Several Paradigms were Considered:

a) A One microRNA-Multiple mRNAs Pathway-Specific Paradigm.

A. The methylation state of histones can be dynamically regulated by histone methyltransferases and demethylases. The human lysine (K)-specific demethylase 3A (KDM3A) is critically important in regulating the expression of metabolic genes. Its loss of function results in obesity and hyperlipidemia in mice. Beta-adrenergic stimulation of KDM3A binding to the PPAR responsive element (PPRE) of the UCP1 gene not only decreases levels of H3K9me2 (dimethylation of lysine 9 of histone H3) at the PPRE, but also facilitates the recruitment of PPARG and RXRA and their co-activators PPARGC1A, CREBBP and NCOA1 to the PPRE. The interrogation of the TargetScan Human database (release 6.0) revealed that the human KDM3A 3′ UTR 29-35 region is a conserved target for hsa-miR-22. Several other miRNA Targets Databases also confirmed this match between hsa-miR-22 and KDM3A. Therefore, increased production of the demethylase KDM3A by an hsa-miR-22 antagomir should lead to demethylation of the UCP1 gene promoter region, thus facilitating binding of several regulatory elements and increased UCP1 production.

In addition, we used the 34 miRNA Targets and Expression tools (Table 2B) to identify the mRNA targets of a given miRNA.

TABLE 2B Bioinformatics tools used to select miRNAs and their targets. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 DIANA-microT 3.0 1 X ✓ DIANA-mirExTra 2 X GOmir 3 X ✓ GSEA MSD v3.0 4 X MAMI 5 ✓ X ✓ ✓ MicroCosm Targets 6 X MicroInspector 7 X microRNA.org 8 X mimiRNA 9 ✓ X MMIA 10 X miRDB 11 X mirDIP 12 ✓ ✓ ✓ X miRGator v3.0 13 ✓ ✓ ✓ X ✓ miRecords 14 ✓ ✓ ✓ X MiRNA Distiller 15 ✓ ✓ X MiRonTop 16 ✓ ✓ X miRror 17 ✓ ✓ ✓ ✓ X miRSystem 18 ✓ ✓ X miRTarBase 19 miRTar.Human 20 miRvestigator 21 miRWalk 22 ✓ ✓ ✓ mirZ 23 MultiMiTar 24 PhenomiR 25 PicTar 26 PITA 27 RepTar 28 RNA22 29 RNAhybrid 30 StarBase 31 ✓ Sylamer 32 TarBase 6.0 33 TargetScanHuman 34 5 4 1 10 3 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 DIANA-microT 3.0 DIANA-mirExTra GOmir ✓ ✓ ✓ 4 GSEA MSD v3.0 MAMI ✓ ✓ 5 MicroCosm Targets MicroInspector microRNA.org mimiRNA ✓ ✓ ✓ 4 MMIA ✓ ✓ ✓ 3 miRDB mirDIP ✓ ✓ ✓ ✓ 7 miRGator v3.0 ✓ ✓ ✓ ✓ ✓ 9 miRecords ✓ ✓ ✓ ✓ ✓ 8 MiRNA Distiller ✓ 3 MiRonTop ✓ ✓ 4 miRror ✓ ✓ ✓ ✓ ✓ 9 miRSystem ✓ ✓ ✓ ✓ 7 miRTarBase X miRTar.Human X miRvestigator X miRWalk X ✓ ✓ ✓ ✓ ✓ 8 mirZ X MultiMiTar X PhenomiR X PicTar X PITA X RepTar X RNA22 X RNAhybrid X StarBase ✓ ✓ ✓ X ✓ 5 Sylamer X TarBase 6.0 X TargetScanHuman X 1 10 8 7 3 12 Meta Tools in bold (13) Engines called by Meta Tools in Italics (11)

Applying the above in silico strategy, it was discovered that hsa-miR-22-3p and hsa-miR-22-5p interact respectively with a total of 42 and 8 of the chosen 83 thermogenic targets. This data is set forth in Table 3.

TABLE 3 Thermogenic regulators identified as predicted and/or validated targets for hsa-miR-22-3p. ALDH1A1 BMP4 BMP7 CEBPA CEBPD CIDEC CREB1 CREBBP CTNNB1 DIO2 FGF19 FGF21 FOXC2 INSR KDM3A KLF11 LRP6 MAPK14 NCOA1 NPPA NRF1 NRIP1 PPARA PPARGC1A PPARGC1B PRDM16 PRDX3 PRKAA1 PRKACA PRKACB PRKAR1A RUNX1T1 RUNX2 SIRT1 SREBF1 SREBF2 STAT5A TNFRSF1A TRPM8 UCP2 WNT10B WNT5A Thermogenic regulators identified as predicted and/or validated targets for hsa-miR-22-5p BMP7 DIO2 FNDC5 IKBKE INSR MAPK14 NR1H3 PPARA

B. We also utilized the 34 miRNA Targets and Expression tools (Table 2B) to look for potential relations between any of the adipocyte 536 miRNAs (Table A) and the 83 thermogenic targets (Table 1).

It appears that many adipocyte miRNAs interact (prediction and/or validation) with at least one of the 83 thermogenic targets. For example, miR-17-3p and hsa-miR-17-5p interact respectively with a total of 23 and 65 of the chosen thermogenic 83 targets. This data is set forth in Table 4.

TABLE 4 Thermogenic regulators identified as predicted and/or validated targets for hsa-miR-17-3p. ATG7 BMP2 BMP4 CEBPB CREB1 CTBP2 E2F4 FGF19 IKBKE IRS1 KLF11 MAPK14 NCOA3 PLAC8 PPARA PPARD PRDM16 RB1 RUNX1T1 STAT5A TNFRSF1A TWIST1 WNT10B Thermogenic regulators identified as predicted and/or validated targets for hsa-miR-17-5p ALDH1A1 ATG7 BMP2 BMP4 BMP7 BMP8b CEBPA CEBPB CEBPD CIDEC COX7A1 CRAT CREB1 CREB2 CTNNB1 CTBP1 CTBP2 DIO2 ELOVL3 FGF19 FGF21 FNDC5 FOXC2 GDF3 HCRT HOXC8 IKBKE INSR IRS1 KLF11 MAPK14 MED13 NCOA1 NCOA2 NCOA3 NPPA NR1H3 NR4A3 NRF1 NRIP1 PLAC8 PPARA PPARD PPARG PPARGC1A PPARGC1B PRDX3 PRKAA1 PRKAA2 PRKACA PRKACB PRKAR1A RB1 RPS6KB1 RUNX1T1 RUNX2 SIRT1 SIRT3 SREBF1 STAT5A TNFRSF1A TWIST1 UCP1 UCP3 WNT5A

Once the lists of miRNAs of interest and their mRNA targets were produced, the following filters were applied to refine the results:

Parameters

1 Expression of miRNAs in tissue/cell of interest

2 Number of algorithms predicting one miRNA for a given gene or set of genes

3 Score/percent from algorithms

4 Number of preferred genes targeted by one miRNA

5 Number of binding sites in a target gene for one miRNA

6 Number of binding sites in a target gene for several miRNAs

7 Over-representation of one miRNA seed complementary sequence among target genes (miRvestigator)

8 Validated miRNA-mRNA target couples

9 Genomic location of miRNA binding site (5′UTR-Promoter-CDS-3′UTR)

10 Intronic location of miRNA

11 Clustering of miRNAs

12 Abundance of miRNA in specific tissue/cell of interest

Applying the above parameters, it was discovered that 229 miRNAs met at least two of these criteria. This data is set forth in Table 5.

TABLE 5 Ranking of miRNAs according to selection critria. hsa-miR-20b-5p hsa-miR-27b-3p hsa-miR-103a-3p hsa-miR-22-3p hsa-miR-34a-5p hsa-miR-130b-3p hsa-miR-132-3p hsa-miR-181b-5p hsa-miR-211-5p hsa-miR-148b-3p hsa-miR-17-5p hsa-miR-182-5p hsa-miR-20a-5p hsa-miR-27a-3p hsa-miR-301a-3p hsa-miR-204-5p hsa-miR-143-3p hsa-miR-1 hsa-miR-9-5p hsa-miR-30a-5p hsa-miR-138-5p hsa-miR-217 hsa-miR-19b-3p hsa-miR-382-5p hsa-miR-106a-5p hsa-miR-107 hsa-miR-135a-5p hsa-miR-93-5p hsa-miR-21-5p hsa-miR-515-3p hsa-miR-106b-3p hsa-miR-125a-5p hsa-miR-148a-3p hsa-miR-155-5p hsa-miR-181a-5p hsa-miR-519d hsa-miR-96-5p hsa-miR-212-3p hsa-miR-29a-3p hsa-miR-98-5p hsa-let-7c hsa-let-7d-5p hsa-miR-141-3p hsa-miR-183-5p hsa-miR-19a-3p hsa-miR-196a-5p hsa-miR-30b-5p hsa-miR-378a-3p hsa-miR-302c-5p hsa-miR-30e-5p hsa-miR-130a-3p hsa-let-7e-5p hsa-miR-216a-5p hsa-miR-450a-5p hsa-let-7d-3p hsa-miR-26b-5p hsa-miR-181c-5p hsa-miR-186-5p hsa-miR-519c-3p hsa-let-7b-5p hsa-miR-10b-5p hsa-miR-125b-5p hsa-miR-134 hsa-miR-137 hsa-miR-150-5p hsa-miR-153 hsa-miR-15b-5p hsa-miR-16-5p hsa-miR-195-5p hsa-miR-196b-5p hsa-miR-23a-3p hsa-miR-29c-3p hsa-miR-373-3p hsa-miR-7-5p hsa-miR-214-3p hsa-miR-421 hsa-miR-15a-5p hsa-miR-193b-3p hsa-miR-194-5p hsa-miR-223-3p hsa-miR-30d-5p hsa-miR-424-5p hsa-miR-454-3p hsa-miR-545-3p hsa-miR-485-5p hsa-miR-335-5p hsa-miR-133a hsa-miR-222-3p hsa-miR-494 hsa-miR-498 hsa-miR-513a-5p hsa-miR-92a-3p hsa-miR-495-3p hsa-miR-503-5p hsa-miR-539-5p hsa-miR-16-2-3p hsa-miR-302b-5p hsa-miR-425-3p hsa-miR-99a-3p hsa-let-7a-3p hsa-miR-126-3p hsa-miR-20a-3p hsa-miR-499a-5p hsa-let-7g-5p hsa-miR-152 hsa-miR-26a-5p hsa-miR-124-3p hsa-miR-203a hsa-miR-24-3p hsa-miR-301b hsa-miR-590-3p hsa-miR-1179 hsa-miR-325 hsa-miR-552 hsa-miR-185-5p hsa-miR-455-3p hsa-miR-583 hsa-miR-122-5p hsa-miR-1305 hsa-miR-139-5p hsa-miR-146a-5p hsa-miR-18a-5p hsa-miR-18b-5p hsa-miR-199b-5p hsa-miR-340-5p hsa-miR-34c-5p hsa-miR-423-3p hsa-miR-489 hsa-miR-520f hsa-miR-520g hsa-miR-605 hsa-miR-668 hsa-let-7a-5p hsa-let-7f-5p hsa-miR-10a-3p hsa-miR-135b-5p hsa-miR-144-3p hsa-miR-181d hsa-miR-200b-3p hsa-miR-200c-3p hsa-miR-218-5p hsa-miR-23b-3p hsa-miR-25-3p hsa-miR-29b-3p hsa-miR-383 hsa-miR-202-3p hsa-miR-381-3p hsa-miR-377-3p hsa-miR-452-5p hsa-miR-501-3p hsa-miR-514a-3p hsa-miR-654-3p hsa-let-7b-3p hsa-miR-125a-3p hsa-miR-133b hsa-miR-192-5p hsa-miR-199a-3p hsa-miR-30c-5p hsa-miR-335-3p hsa-miR-374a-5p hsa-miR-410 hsa-miR-429 hsa-miR-497-5p hsa-miR-513a-3p hsa-miR-542-3p hsa-miR-653 hsa-miR-122-3p hsa-miR-101-5p hsa-miR-1178-3p hsa-miR-191-5p hsa-miR-214-5p hsa-miR-302d-5p hsa-miR-572 hsa-miR-574-3p hsa-miR-26a-2-3p hsa-miR-611 hsa-let-7f-1-3p hsa-let-7i-3p hsa-miR-100-5p hsa-miR-106b-5p hsa-miR-132-5p hsa-miR-135b-3p hsa-miR-136-3p hsa-miR-150-3p hsa-miR-154-3p hsa-miR-15a-3p hsa-miR-15b-3p hsa-miR-16-1-3p hsa-miR-181a-2-3p hsa-miR-181c-3p hsa-miR-186-3p hsa-miR-195-3p hsa-miR-20b-3p hsa-miR-223-5p hsa-miR-224-3p hsa-miR-24-1-5p hsa-miR-24-2-5p hsa-miR-27a-5p hsa-miR-27b-5p hsa-miR-29b-1-5p hsa-miR-302a-5p hsa-miR-3065-5p hsa-miR-30d-3p hsa-miR-34a-3p hsa-miR-371a-3p hsa-miR-373-5p hsa-miR-374a-3p hsa-miR-376a-5p hsa-miR-378a-5p hsa-miR-424-3p hsa-miR-451a hsa-miR-452-3p hsa-miR-487b hsa-miR-493-5p hsa-miR-500a-3p hsa-miR-502-3p hsa-miR-516b-3p hsa-miR-518e-3p hsa-miR-518f-3p hsa-miR-519a-5p hsa-miR-519b-5p hsa-miR-521 hsa-miR-523-5p hsa-miR-545-5p hsa-miR-585 hsa-miR-7-2-3p hsa-miR-93-3p hsa-miR-96-3p hsa-miR-99b-3p c) A Multiple microRNAs-One mRNA Paradigm.

A. One exemplary multiple miRNAs-one mRNA paradigm involves UCP1. In adipocytes the key thermogenic regulator ultimately is UCP1 (also named thermogenin) and, thus, all thermogenic regulators must ultimately impact UCP1 activity. UCP1 is a mitochondrial transporter protein that creates proton leaks across the inner mitochondrial membrane, thus uncoupling oxidative phosphorylation from ATP synthesis. As a result, energy is dissipated in the form of heat (adaptive thermogenesis) (see FIG. 5) Lowell et al., Nature (2000); Friedman et al., Bioinformatics (2010); Hsu et al., Nucleic acids research (2011); Rieger et al., Frontiers in Genetics (2011)).

UCP1 biosynthesis is mainly controlled at the transcription level. FIG. 6 depicts the transcriptional control of UCP1 by other exemplary thermogenic regulators. The promoter's region of the UCP1 gene contains many distinct regulatory sites, allowing a wide range of proteins to influence its transcription, both positively (see FIG. 7A) and negatively (see FIG. 7B).

Mendelian randomization is a method of using measured variation in genes of known function to examine the causal effect of a modifiable exposure on disease in non-experimental studies. Mendelian randomization can be thought of as a “natural” Randomized Clinical Trial. Genetic polymorphism of the UCP1 gene, such as the −3826 A/G single nucleotide polymorphism in the promoter in exon 2 of UCP1, has been reported to be associated with reduced mRNA expression and obesity. Healthy children with the G/G genotype had a lower capacity for thermogenesis in response to a high-fat meal and acute cold exposure. The same −3826 A/G UCP1 genetic polymorphism diminishes resting energy expenditure and thermoregulatory sympathetic nervous system activity in young females. In a study of 367 Korean women, the G allele of −3826A>G and the C allele of −412A>C were significantly associated with larger areas of abdominal subcutaneous fat in a dominant model (p<0.001 and p<0.0004, respectively); combining them together (ht2[GC]) enhanced this significance (p<0.00005). A study of 100 severe obese adults (BMI>40 kg/m2) and 100 normal-weight control subjects (BMI range=19-24.9 kg/m2) identified 7 variations in the promoter region, 4 in the intronic region and 4 in the exonic region of the UCP1 gene. These variations could contribute to the development of obesity, particularly, g.−451C>T, g.940G>A, and g.IVS4-208T>G could represent “thrifty” factors that promote energy storage. Finally, two polymorphisms (A-3826G and C-3740A), located in the upstream promoter region of the UCP1 gene affect gene expression and are correlated with human longevity.

All aforementioned information supports targeting UCP1 expression and activity as a meaningful way to alter adaptive thermogenesis and consequently treat human obesity. Many strategies could be implemented to achieve this goal, however, the one employed in the methods of the invention uses miRNA agents to modulate simultaneously several elements within the thermogenic pathways to increase UCP1 synthesis and activity. Both direct and indirect interactions between miRNAs and the UCP1 gene are considered. Direct interaction means the direct binding of miRNAs to the various regions of the UCP1 gene, resulting in alterations of the transcription, translation, stability and/or degradation of the UCP1 mRNA. Indirect interaction means that miRNAs alter the transcription, translation, stability and/or degradation of thermogenic mRNAs, whose expressed proteins alter the transcription of the UCP1 gene. Furthermore, indirect interaction means that miRNAs alter the transcription, translation, stability and/or degradation of other miRNAs that modify the transcription of the UCP1 gene.

The promoter region of the human UCP1 gene (gi|237858805|ref|NG_012139.1| Homo sapiens uncoupling protein 1 (mitochondrial, proton carrier) (UCP1), RefSeqGene on chromosome 4) is particularly rich is regulatory element motifs:

UCP1 Gene Regulatory Elements: 1. Brown Fat Response Element 1 (BRE1) Motif: CCTCTCTGCTTCTTCT [SEQ ID NO: 1] One Length: 16, Interval: 1,129->1,144, Mismatches: 0. 2. Brown Fat Response Element 2 (BRE2) Motif: CTCCTTGGAA [SEQ ID NO: 2] One Length: 10, Interval: 1,269->1,278, Mismatches: 0. 3. CRE2 Motif: ATTCTTTA Four Length: 8, Intervals: 1,121->1,128, 3,631->3,638, 10,982->10,989, 15,881->15,888, Mismatches: 0. 4. CREB Motif: ACGTCA Five Length: 6, Intervals: 1,082->1,087, 1,345->1,350, 1,348->1,343, 11,439->11,434, 13,831->13,836, Mismatches: 0. 5. DR1 Motif: TTGCCCTTGCTCA [SEQ ID NO: 3] One Length: 13, Interval: 1,099->1,111, Mismatches: 0. 6. DR4 Motif: ACGTCATAAAGGGTCA [SEQ ID NO: 4] One Length: 16, Interval: 1,082->1,097, Mismatches: 0. 7. DR4 Type RARE Motif: RGKTCANNNNRGKTCA [SEQ ID NO:5] One Length: 16, Interval: 1,316->1,301, Mismatches: 0. 8. ERE Motif: GCTCATACTGACCT [SEQ ID NO:6] One Length: 14, Interval: 1,107->1,120, Mismatches: 0. 9. PRE Motif: GTTAATGTGTTCT [SEQ ID NO:7] One Length: 13, Interval: 1,009->1,021, Mismatches: 0. 10. RARE Motif: TGACCACAGTTTGATCA [SEQ ID NO: 8] One Length: 17, Interval: 983->999, Mismatches: 0. 11. RXR Motif: AGGTCA Twelve Length: 6, Interval: 1,120->1,115, 1,316->1,311, 3,517->3,522, 3,560->3,555, 3,813->3,808, 5,318->5,313, 6,233->6,238, 6,831->6,836, 8,122->8,127, 9,966->9,971, 11,339->11,334, 11,412->11,407, Mismatches: 0. 12. GC Box 1 Motif: CGCCC Seven Length: 5, Interval: 4,593->4,589, 4,615->4,619, 4,615->4,619, 4,747->4,751, 4,765->4,769, 5,914->5,910, 13,715->13,711, Mismatches: 0. 13. GC Box 2 Motif: GCGGG Nine Length: 5, Interval: 4,463->4,459, 4,585->4,589, 4,593->4,597, 4,639->4,643, 4,883->4,887, 5,176->5,172, 5,929->5,933, 5,940->5,944,14,994->14,990, Mismatches: 0. 14. GT Box 1 Motif: CACCC Twenty Five

Length: 5, Interval: 194->190, 452->448, 1,184->1,188, 1,803->1,807, 2,428->2,424, 3,037->3,041, 3,330->3,334, 4,137->4,141, 4,566->4,562, 4,599->4,595, 4,869->4,865, 5,104->5,108, 5,461->5,457, 6,237->6,241, 6,293->6,289, 8,096->8,092, 8,198->8,194, 9,649->9,645, 9,912->9,908, 12,962->12,958, 13,136->13,132, 13,723->13,719, 14,404->14,400, 14,960->14,964, 15,576->15,572, Mismatches: 0.

15. GT Box 2 Motif: GTGGG Twenty

Length: 5, Interval: 25->21, 1,805->1,801, 1,809->1,805, 2,119->2,123, 3,854->3,850, 4,310->4,314, 4,339->4,343, 4,765->4,761, 4,867->4,871, 6,291->6,295, 7,554->7,558, 8,280->8,284, 8,681->8,685, 9,615->9,619, 9,689->9,693, 9,906->9,910, 10,363->10,359, 13,074->13,070, 13,640->13,644, 13,941->13,945, Mismatches: 0.

16. CpG Methylation Island Motif: CG

Three Hundred and Sixty Six, including many between positions 4,519 to 5,258 and 5,639 to 6,694.

FIG. 8A depicts the location of these various regulatory elements in reference to the UCP1 transcription start site at nucleotide position 5,001 of the 15,910 base pair human UCP-1 gene (FASTA accession number: >gi|237858805|ref|NG_012139.11 Homo sapiens uncoupling protein 1 (mitochondrial, proton carrier) (UCP1), RefSeqGene on chromosome 4; NCBI Reference Sequence: NG_012139.1).

Direct or indirect activation or repression of these regulatory elements by miRNAs will result in alterations of UCP1 gene expression and activity. Under normal conditions, the UCP1 gene expression and activity are repressed by a rich network of regulatory elements, in order to avoid energy wasting. Under stress, such as exposure to a cold environment, the expression of the UCP1 gene is upregulated, via various activators and repressors which are under the control of several miRNAs.

An initial survey of miRNAs targeting the human UCP1 3′UTR with several programs, including microRNA.org, was negative. However, other programs, including MicroCosm Targets, using the UCP1 Ensembl 1,462 base pair transcript ENST00000262999 as a target revealed binding sites for 27 miRNAs at 28 locations in UCP1 3′UTR as shown in Table 6.

TABLE 6 Binding sites for miRNAs in the 3′UTR of UCP1 (NCBI Reference Sequence. NG_012139.1) determined using microCosm Targets. SEQ ID Name Sequence NO Minimum Maximum Length hsa-miR-21 AATGTAATGCAGATAAGCTA  9 14143 14162 20 hsa-miR-219-2-3p ACATGTTTTAATTACAATTC 10 14217 14236 20 hsa-miR-22 GATTGGCAGCTT 11 14857 14868 12 hsa-miR-222a GATTTTTAATGTTTAGAGTCCAG 12 14500 14522 23 hsa-miR-290-3p TTTAGAGCTGGAGGGTACTT 13 14621 14640 20 hsa-miR-292-3p TTTAGAGCTGGAGGGTACTT 14 14621 14640 20 hsa-miR-292-5p GACAGAGGAACAGTTTGAG 15 14648 14666 19 hsa-miR-325 ATTTTGGCAGGATTGCTACTAG 16 14568 14589 22 hsa-miR-331-5p TTTTGAGATCTATACCTGG 17 14383 14401 19 hsa-miR-362-5p ATTTTAAGCTAAATCCAAGGATT 18 14838 14860 23 hsa-miR-367 TGACCATTTCTGGAGTGCAATT 19 14170 14191 22 hsa-miR-371-5p ACAGTTTGAT 20   988   997 10 hsa-miR-371-5p ACAGTTTGAG 21 14657 14666 10 hsa-miR-377 CTGGAGTGCAATTGTGTGA 22 14179 14197 19 hsa-miR-378 TTTTAATGTTTAGAGTCCAG 23 14503 14522 20 hsa-miR-382 TGATGACATCTCTAACAACTTC 24 14526 14547 22 hsa-miR-460 AGAAACTGAGTGAAATGCAG 25 14250 14269 20 hsa-miR-508-5p TGACCATTTCTGGAGTG 26 14170 14186 17 hsa-miR-543 TACTCTGAATGTT 27 14478 14490 13 hsa-miR-549 TTAACCACAGTTGTCA 28 14321 14336 16 hsa-miR-643 CAAGTTCACTAGAATACAAG 29 14412 14431 20 hsa-miR-654-3p AAGGTTACAGGCTGCCAGACAT 30 14880 14901 22 hsa-miR-664 GTGTGAATGAATG 31 14192 14204 13 hsa-miR-871 TAGGCATGAACCTACTCTGAATG 32 14466 14488 23 hsa-miR-883a-3p AAACTGAGTGAAATGCAGTT 33 14252 14271 20 hsa-miR-883b-3p AAACTGAGTGAAATGCAGTT 34 14252 14271 20 hsa-miR-888-3p TTTATTAACCACAGTTGTCAGTT 35 14317 14339 23 hsa-miR-92b GAGTGCAAT 14182 14190  9

Other programs, such as miRWalk, miRGen, miRGator-miRanda, and DIANA microT, using the UCP1 Ensembl 1,462 base pair transcript (ENST00000262999), the UCP1 Ensembl 9,371 base pair gene sequence (ENSG00000109424) or the 15,910 base pair UCP1 sequence (NCBI Reference Sequence: NG_012139.1) as targets, revealed binding sites for a total of 50 miRNAs at 69 locations in UCP1 3′UTR as shown in Table 7.

TABLE 7 Binding sites for miRNAs in the 3′UTR of UCP1 (NCBI Reference Sequence. NG_012139.1) according to several programs. SEQ ID Name Sequence NO Minimum Maximum Length  1 hsa-miR-1179 AAGTATCCTTT 36 15346 15356 11  2 hsa-miR-1302 ATGGGACACA 37 15021 15030 10  3 hsa-miR-130b TTATTTTCCCT 38 15161 15171 11  4 hsa-miR-146a TGACAACTGT 39 14327 14336 10 hsa-miR-146a AGGGAACTGA 40 15231 15240 10 hsa-miR-146a TGTGAACTGG 41 15679 15688 10  5 hsa-miR-181c AACCATAGT 15304 15312  9  6 hsa-miR-19b-2 ACTTTTGCGG 42 14991 15000 10  7 hsa-miR-203 TTAAATGTT 15584 15592  9  8 hsa-miR-204-5p TTCCTTTATC 43 14006 14015 10 hsa-miR-204-5p TTCCTCTGTC 44 14648 14657 10  9 hsa-miR-21-5p TAGCTTATCT 45 14153 14162 10 10 hsa-miR-211-5p TTCCCTATCTC 46 14779 14789 11 11 hsa-miR-214 CAGCAAGCA 47 15052 15060  9 12 hsa-miR-22-3p AAGCTGCCAA 48 14859 14868 10 hsa-miR-22-5p AGTTCTTCACA 49 14203 14213 11 13 hsa-miR-26a-2-3p CATTTTCTTG 50 13918 13927 10 hsa-miR-26a-2-3p CCAATCCTTG 51 14853 14862 10 hsa-miR-26a-2-3p CCTTTTCATG 52 15616 15625 10 14 hsa-miR-30b GTAACCTTCC 53 14878 14887 10 15 hsa-miR-325 CAGAGTAGGT 54 14475 14484 10 hsa-miR-325 CCTTGTAGGC 55 15378 15387 10 16 hsa-miR-328 CTGTTCCTCT 56 14651 14660 10 17 hsa-miR-362-5p ATCCTTGGAT 57 14850 14859 10 18 hsa-miR-367-3p AATTGCACTC 58 14182 14191 10 19 hsa-miR-371a-3p AAGTGCCTGC 59 15435 15444 10 hsa-miR-371a-5p TCTCAAACTG 60 14658 14667 10 20 hsa-miR-378a-3p ACTGGCCTTG 61 15816 15825 10 21 hsa-miR-382-3p ATTCATTCAC 62 14194 14203 10 22 hsa-miR-382-5p GAAGTTGTTAGAGAT 63 14533 14547 15 23 hsa-miR-383 AGATTAGAA 64 14545 14553  9 24 hsa-miR-421 ATTAACTGAC 65 14333 14342 10 hsa-miR-421 CTCAAAAGAC 66 14380 14389 10 25 hsa-miR-422a ACTGGCCTT 15817 15825  9 26 hsa-miR-431 TGTCTGGCA 14892 14900  9 27 hsa-miR-452 TTATCTGC 14151 14158  8 hsa-miR-452 TCTTCTGC 14773 14780  8 hsa-miR-452 ACATCTGC 15009 15016  8 28 hsa-miR-455-3p CAGTCCAT 13893 13900  8 hsa-miR-455-5p TGTGTGCCTT 67 15641 15650 10 29 hsa-miR-491-5p AATGGGGAAG 68 14975 14984 10 30 hsa-miR-501-3p ATGCATCAGG 69 15547 15556 10 31 hsa-miR-504 AGACCCTGT 15325 15333  9 TATTCTAGTGAACTTG 70 32 hsa-miR-508-5p ACTCTTA 14405 14427 23 33 hsa-miR-512-5p CACTCAG 14255 14261  7 34 hsa-miR-514a-3p TTGACTCTT 14406 14414  9 35 hsa-miR-515-3p GACTGCCTT 15539 15547  9 hsa-miR-515-3p GTGTGCCTT 15641 15649  9 36 hsa-miR-517a-3p ATGGTGCATT 71 15650 15659 10 37 hsa-miR-545 CAGCAAGCACT 72 15050 15060 11 38 hsa-miR-549 TGACAACTGT 73 14327 14336 10 39 hsa-miR-552 CACAGGTGA 15130 15138  9 40 hsa-miR-616-5p ACTCTAAAC 14510 14518  9 41 hsa-miR-620 ATGAATATAG 74 14560 14569 10 42 hsa-miR-643 ACTGGTATGT 75 13933 13942 10 hsa-miR-643 TCTTGTATTC 76 14423 14432 10 hsa-miR-643 CCTTGTAGGC 77 15378 15387 10 hsa-miR-643 ACATGCATGC 78 15553 15562 10 43 hsa-miR-651 TTAAAATAAG 79 13988 13997 10 hsa-miR-651 TTAGGTTAAA 80 13993 14002 10 hsa-miR-651 TCATGATAAG 81 15700 15709 10 44 hsa-miR-654-3p TATCTCTTCT 82 14775 14784 10 hsa-miR-654-3p TATGTATACT 83 15493 15502 10 45 hsa-miR-655 GTAATACAT 15593 15601  9 46 hsa-miR-767-3p CCTGCTCAT 14871 14879  9 47 hsa-miR-888-3p GACTGACTCC 84 15772 15781 10 48 hsa-miR-92b-3p ATTGCACTCC 85 14181 14190 10 49 hsa-miR-941 CACCCAGGT 14396 14404  9 50 hsa-miR-99a-3p AAGCTGGCTC 86 15117 15126 10

Alignment of the sequence of the human UCP1 gene with several miRNA sequences yielded matches in the 5′UTR, the promoter region and the coding regions of the UCP1 gene. Interrogation of the publicly available Internet tools predicting miRNAs targeting the various regions of the UCP1 gene elicited several hits. Surprisingly, the overlap between these prediction tools was zero, as shown in FIG. 3.

Nevertheless, miRNA databases were screened using the alignment program Geneious. A total of 191 human microRNAs were found which have complementary 450 binding sites in the UCP1 gene sequence (Table 8). The length of the matches goes from 7 bases to 12 bases (e.g. hsa-miR-24-2-5p and hsa-miR-192-5p). The number of hits per miRNA varies from 1 to several (e.g. 9 for hsa-miR-19b2 (an abundant adipocyte miRNA), 14 for hsa-miR-26a-2-3p, 11 for hsa-miR-181c, and 12 for hsa-miR-620).

TABLE 8 miRNAs with predicted binding sites in the UCP1 gene sequence (NCBI Reference Sequence: NG_012139.1). SEQ ID miRNA Sequence NO Minimum Maximum Length Direction hsa-let-7c TAGAGTTTC 5918 5926 9 reverse hsa-let-7e GGAGGTAGG 13283 13291 9 reverse hsa-let-7e TGAAGTAGG 7612 7620 9 reverse hsa-let-7e AGAGGTAGG 3306 3314 9 reverse hsa-let-7i-3p CTGTGCAAG 3588 3596 9 reverse hsa-miR-17 CAAAGTGCT 12200 12208 9 reverse hsa-miR-17 CAAAGTGCT 9931 9939 9 reverse hsa-miR-17 CAAAGTGCT 218 226 9 reverse hsa-miR-19a TGTGCAAAT 3916 3924 9 reverse hsa-miR-19a TGTGCAAAT 834 842 9 reverse hsa-miR-19b-2 ACTTTTGCGG  87 14991 15000 10 reverse hsa-miR-19b-2 AGTTTTACAA  88 11998 12007 10 reverse hsa-miR-19b-2 AGTTTTGTAT  89 10023 10032 10 reverse hsa-miR-19b-2 AGTCTTGAAG  90 9399 9408 10 reverse hsa-miR-19b-2 AGGTTTGTAG  91 7758 7767 10 reverse hsa-miR-19b-2 AGTATTGAAG  92 7159 7168 10 reverse hsa-miR-19b-2 AGGCTTGCAG  93 3546 3555 10 reverse hsa-miR-19b-2 AATTTGGCAG  94 529 538 10 reverse hsa-miR-19b-2 AGTTTTGGAA  95 312 321 10 reverse hsa-miR-20b CAAAGTGCT 12200 12208 9 reverse hsa-miR-20b CAAAGTGCT 9931 9939 9 reverse hsa-miR-20b CAAAGTGCT 218 226 9 reverse hsa-miR-21-5p TAGCTTATCT  96 14153 14162 10 reverse hsa-miR-22-3p AAGCTGCCAA  97 14859 14868 10 reverse hsa-miR-22-3p AAGCTTCCAG  98 1482 1491 10 reverse hsa-miR-22-5p AGTTCTTCACA  99 14203 14213 11 reverse hsa-miR-22-5p AATTCTTCAGG 100 8032 8042 11 reverse hsa-miR-22-5p GGTTCTTCAGC 101 5389 5399 11 reverse hsa-miR-24-2-5p TGCCTACTGGCC 102 8651 8662 12 reverse hsa-miR-25-3p CATTGCAC 11565 11572 8 reverse hsa-miR-25-5p AGGCGGAG 5963 5970 8 reverse hsa-miR-26a-2-3p CCTTTTCATG 103 15616 15625 10 reverse hsa-miR-26a-2-3p CCAATCCTTG 104 14853 14862 10 reverse hsa-miR-26a-2-3p CATTTTCTTG 105 13918 13927 10 reverse hsa-miR-26a-2-3p CCTACTCTTC 106 13505 13514 10 reverse hsa-miR-26a-2-3p ACGATTCTTG 107 13192 13201 10 reverse hsa-miR-26a-2-3p TCTATTCTTT 108 12883 12892 10 reverse hsa-miR-26a-2-3p CATATTTTTG 109 10197 10206 10 reverse hsa-miR-26a-2-3p GCTAGTCTTG 110 9978 9987 10 reverse hsa-miR-26a-2-3p CATATTTTTG 111 9890 9899 10 reverse hsa-miR-26a-2-3p CCTTTTCTTT 112 6631 6640 10 reverse hsa-miR-26a-2-3p CCCATTCTCG 113 4709 4718 10 reverse hsa-miR-26a-2-3p TTTATTCTTG 114 3893 3902 10 reverse hsa-miR-26a-2-3p CCTTTACTTG 115 1885 1894 10 reverse hsa-miR-26a-2-3p GCGATTCTTG 116 376 385 10 reverse hsa-miR-27-5p AGAGCTTAGG 117 2949 2958 10 reverse hsa-miR-30b GTAACCTTCC 118 14878 14887 10 reverse hsa-miR-30b GTAACCATCA 119 12991 13000 10 reverse hsa-miR-30b GTAATCATAC 120 12831 12840 10 reverse hsa-miR-30b GTCAACATCA 121 11401 11410 10 reverse hsa-miR-30b GTAAACATAA 122 9365 9374 10 reverse hsa-miR-30b GTACTCATCC 123 9016 9025 10 reverse hsa-miR-30b CTATACATCC 124 8586 8595 10 reverse hsa-miR-30b CTAAACATCT 125 7495 7504 10 reverse hsa-miR-31 GGCTATGCC 7712 7720 9 reverse hsa-miR-32 ATTGCACA 11564 11571 8 reverse hsa-miR-92b ATTGCACTCC 126 14181 14190 10 reverse hsa-miR-92b ATTGCACTAG 127 11282 11291 10 reverse hsa-miR-93 CAAAGTGCTG 128 12199 12208 10 reverse hsa-miR-93 CAAAGTGCTG 129 217 226 10 reverse hsa-miR-93-3p ACTCCTGGGCT 130 12356 12366 11 reverse hsa-miR-93-3p ACTGATAAGCT 131 11055 11065 11 reverse hsa-miR-93-3p ACTCCTGACCT 132 9966 9976 11 reverse hsa-miR-96-3p AATCATGTGCC 133 8659 8669 11 reverse hsa-miR-99a-3p AAGCTGGCTC 134 15117 15126 10 reverse hsa-miR-99a-3p AAACTCTTTC 135 13344 13353 10 reverse hsa-miR-99a-3p AATCTTGTTC 136 11952 11961 10 reverse hsa-miR-99a-3p AAGCTCCTTT 137 11050 11059 10 reverse hsa-miR-99a-3p AAGCTCCTTT 138 8099 8108 10 reverse hsa-miR-99a-3p AAGCTCTGTC 139 7523 7532 10 reverse hsa-miR-99b-3p CAACCTCGAG 140 13666 13675 10 reverse hsa-miR-99b-3p CGAGCTCCTG 141 13660 13669 10 reverse hsa-miR-99b-3p GAAGCTTGTG 142 6436 6445 10 reverse hsa-miR-99b-3p CAAACTCCTG 143 257 266 10 reverse hsa-miR-100 TCCAGTAGAT 144 11866 11875 10 reverse hsa-miR-100 ACGCGCAGAT 145 5634 5643 10 reverse hsa-miR-106b-5p CAAAGTGCTG 146 12199 12208 10 reverse hsa-miR-106b-5p CAAAGTGCTG 147 217 226 10 reverse hsa-miR-126-3P TCATACAGT 12828 12836 9 reverse hsa-miR-126-3P TTGTACTGT 11542 11550 9 reverse hsa-miR-126-3P TGGTCCCGT 7922 7930 9 reverse hsa-miR-126-3P TCATACAGT 932 940 9 reverse hsa-miR-130b TTATTTTCCCT 148 15161 15171 11 reverse hsa-miR-130b CTCTTTTCAGT 149 9670 9680 11 reverse hsa-miR-130b CTCTCTTCACT 150 8977 8987 11 reverse hsa-miR-130b CTCTTTTTCCC 151 8444 8454 11 reverse hsa-miR-130b CTTTTTCCCCT 152 6624 6634 11 reverse hsa-miR-130b CTATTTTCCGT 153 5742 5752 11 reverse hsa-miR-130b TTCCTTTCCCT 154 5007 5017 11 reverse hsa-miR-130b CTCTTTGCCCC 155 1845 1855 11 reverse hsa-miR-130b CTCCTTTCCTT 156 1033 1043 11 reverse hsa-miR-133a-1 TTTGGTGCCC 157 7393 7402 10 reverse hsa-miR-140-3p TACCACAG 5893 5900 8 reverse hsa-miR-141 TAACACTG 5852 5859 8 reverse hsa-miR-143 GGTGCAGTG 158 4132 4140 9 reverse hsa-miR-143-3p TGAGATGAGG 159 13727 13736 10 reverse hsa-miR-143-3p TGAGATGGAG 160 10172 10181 10 reverse hsa-miR-143-3p TTAGATGAAG 161 9572 9581 10 reverse hsa-miR-144-3p TACAGTATT 12825 12833 9 reverse hsa-miR-144-3p TACAATATA 8859 8867 9 reverse hsa-miR-144-3p GACAGTATA 1491 1499 9 reverse hsa-miR-146a CCTCTGAAA 3499 3507 9 reverse hsa-miR-146a TGTGAACTGG 162 15679 15688 10 reverse hsa-miR-146a AGGGAACTGA 163 15231 15240 10 reverse hsa-miR-146a TGACAACTGT 164 14327 14336 10 reverse hsa-miR-146a TAAGAACTAA 165 8935 8944 10 reverse hsa-miR-146a TTAGAACAGA 166 7908 7917 10 reverse hsa-miR-146a TGAGAAGTGC 167 6926 6935 10 reverse hsa-miR-146a TGAAAACTTA 168 3883 3892 10 reverse hsa-miR-146a ACAGAACTGA 169 2259 2268 10 reverse hsa-miR-146a TGAGACCAGA 170 2235 2244 10 reverse hsa-miR-146a TGAGAAATAA 171 1614 1623 10 reverse hsa-miR-147 TGTGTGGATAA 172 7223 7233 11 reverse hsa-miR-147 TTTGTGCAAAT 173 3916 3926 11 reverse hsa-miR-154 AATCATACA 12830 12838 9 reverse hsa-miR-154 AATCATACA 934 942 9 reverse hsa-miR-181c AACCATAGT 15304 15312 9 reverse hsa-miR-181c AACCAAAGA 13244 13252 9 reverse hsa-miR-181c AACCATCAC 12990 12998 9 reverse hsa-miR-181c ATCCAGCGA 11466 11474 9 reverse hsa-miR-181c AAACATCTA 7494 7502 9 reverse hsa-miR-181c AAAAATCGA 6201 6209 9 reverse hsa-miR-181c AACCCCCGA 5540 5548 9 reverse hsa-miR-181c AACCCTCTA 3614 3622 9 reverse hsa-miR-181c AGCCAGCGA 3471 3479 9 reverse hsa-miR-181c AACCATAGG 2801 2809 9 reverse hsa-miR-181c AACCATCAC 194 202 9 reverse hsa-miR-185 TGGAGAGAA 2979 2987 9 reverse hsa-miR-192-5p CTAACATATGAA 174 114 125 12 reverse hsa-miR-194-1 TGTAACAGCA 175 1895 1904 10 reverse hsa-miR-196a AGGTAGTTT 12139 12147 9 reverse hsa-miR-199a-5p CCCTGTGTTC 176 5753 5762 10 reverse hsa-miR-200a TAACACTG 5852 5859 8 reverse hsa-miR-200b TAATAATGCC 177 11184 11193 10 reverse hsa-miR-200b GAATACTGCC 178 10340 10349 10 reverse hsa-miR-200c-3p TAATACTGT 12466 12474 9 reverse hsa-miR-200c-3p TAATAATGC 11185 11193 9 reverse hsa-miR-200c-3p GAATACTGC 10341 10349 9 reverse hsa-miR-200c-3p TAATACAGC 7594 7602 9 reverse hsa-miR-203 TTAAATGTT 15584 15592 9 reverse hsa-miR-203 TGAAATTTT 9782 9790 9 reverse hsa-miR-203 TGAAAGGTT 4495 4503 9 reverse hsa-miR-204-5p TTCCTCTGTC 179 14648 14657 10 reverse hsa-miR-204-5p TTCCTTTATC 180 14006 14015 10 reverse hsa-miR-205 TCCTTCATT 10659 10667 9 reverse hsa-miR-208b ATAAGAAGA 9493 9501 9 reverse hsa-miR-208b ATAAGAAGA 1770 1778 9 reverse hsa-miR-211-5p TTCCCTATCTC 181 14779 14789 11 reverse hsa-miR-211-5p TCCCCTCTGTC 182 5238 5248 11 reverse hsa-miR-211-5p TTCCCTTGCTC 183 5002 5012 11 reverse hsa-miR-211-5p TTCCCATTCTC 184 4710 4720 11 reverse hsa-miR-214 CAGCAAGCA 15052 15060 9 reverse hsa-miR-214 CAGAAGGCA 6918 6926 9 reverse hsa-miR-214 CCGCAGGCA 5935 5943 9 reverse hsa-miR-214 CACCAGGCA 2087 2095 9 reverse hsa-miR-218 TGTGCTTGA 10385 10393 9 reverse hsa-miR-302c TTTAACATG 2932 2940 9 reverse hsa-miR-324-5p CGCGTCCCCT 185 4876 4885 10 reverse hsa-miR-325 CCTTGTAGGC 186 15378 15387 10 reverse hsa-miR-325 CAGAGTAGGT 187 14475 14484 10 reverse hsa-miR-325 CCAAGTAGCT 188 10066 10075 10 reverse hsa-miR-325 CCAAGTAGCT 189 354 363 10 reverse hsa-miR-328 CTGTTCCTCT 190 14651 14660 10 reverse hsa-miR-328 CTGGCTCCCT 191 8215 8224 10 reverse hsa-miR-328 CTGGCCCTTC 192 8062 8071 10 reverse hsa-miR-328 CTGGCACTCA 193 6653 6662 10 reverse hsa-miR-328 CTGGCTTTCT 194 6496 6505 10 reverse hsa-miR-328 CTGCCCCTCC 195 6048 6057 10 reverse hsa-miR-328 CTGGGCCGCT 196 4804 4813 10 reverse hsa-miR-328 CTGGAGCTCT 197 4477 4486 10 reverse hsa-miR-328 CTGACCCTTT 198 1089 1098 10 reverse hsa-miR-330 CAAAGCACAC 199 13845 13854 10 reverse hsa-miR-330 CAAAGCACAC 199 11657 11666 10 reverse hsa-miR-331-5p CTAGGTGTGG 200 7719 7728 10 reverse hsa-miR-361-3p CCCCCAGG 5112 5119 8 reverse hsa-miR-362-5p ATCCTTGGAT 201 14850 14859 10 reverse hsa-miR-367-3p AATTGCACTC 202 14182 14191 10 reverse hsa-miR-367-3p AAATGCACTT 203 999 1008 10 reverse hsa-miR-369 AATAATACA 2266 2274 9 reverse hsa-miR-371a-3p AAGTGCCTGC 204 15435 15444 10 reverse hsa-miR-371a-3p AAGAGCCGAC 205 11455 11464 10 reverse hsa-miR-371a-3p ACGTGCCACC 206 10044 10053 10 reverse hsa-miR-371a-3p AAGTGCCTCT 207 7047 7056 10 reverse hsa-miR-371a-3p AAGTGCACCC 208 5457 5466 10 reverse hsa-miR-371a-5p TCTCAAACTG 209 14658 14667 10 reverse hsa-miR-372 AAAGTGCTG 12199 12207 9 reverse hsa-miR-372 AAAGTGCTG 217 225 9 reverse hsa-miR-374a-3p TCATCAGATT 210 10606 10615 10 reverse hsa-miR-377-3p AGCACACAAA 211 13842 13851 10 reverse hsa-miR-378a-3p ACTGGCCTTG 212 15816 15825 10 reverse hsa-miR-378a-3p ACTGGTCTTG 213 11837 11846 10 reverse hsa-miR-378a-5p CTCCTGCCTC 214 12216 12225 10 reverse hsa-miR-378a-5p CTCCTGCCTC 215 10082 10091 10 reverse hsa-miR-378a-5p CTCCTGTCTC 216 8207 8216 10 reverse hsa-miR-378a-5p CTCCTAACTC 217 7650 7659 10 reverse hsa-miR-382-3p ATTCATTCAC 218 14194 14203 10 reverse hsa-miR-383 AGATTAGAA 14545 14553 9 reverse hsa-miR-383 AGATTAGAA 7912 7920 9 reverse hsa-miR-383 AGAACAGAA 5801 5809 9 reverse hsa-miR-412 ACTTCACCT 737 745 9 reverse hsa-miR-421 CTCAAAAGAC 219 14380 14389 10 reverse hsa-miR-421 ATTAACTGAC 220 14333 14342 10 reverse hsa-miR-421 AACATCAGAC 221 11398 11407 10 reverse hsa-miR-421 ATCAACTGAG 222 3427 3436 10 reverse hsa-miR-421 ATCAACAGGT 223 2443 2452 10 reverse hsa-miR-421 ATCAAAAGAT 224 2333 2342 10 reverse hsa-miR-422a ACTGGCCTT 15817 15825 9 reverse hsa-miR-422a ACTGGTCTT 11838 11846 9 reverse hsa-miR-422a ACTGGACGT 5847 5855 9 reverse hsa-miR-425 AGCGGGAAGGT 225 5167 5177 11 reverse hsa-miR-431 TGTCTGGCA 14892 14900 9 reverse hsa-miR-431 TGTCTAGCA 9218 9226 9 reverse hsa-miR-432-5p TCCTGGAGT 13624 13632 9 reverse hsa-miR-432-5p TATTGGAGT 10785 10793 9 reverse hsa-miR-432-5p TCTTAGAGT 9263 9271 9 reverse hsa-miR-432-5p TCTTAGAGT 6666 6674 9 reverse hsa-miR-432-5p TCTTGGAGC 2180 2188 9 reverse hsa-miR-452 ACATCTGC 15009 15016 8 reverse hsa-miR-452 TCTTCTGC 14773 14780 8 reverse hsa-miR-452 TTATCTGC 14151 14158 8 reverse hsa-miR-452 TCCTCTGC 13488 13495 8 reverse hsa-miR-452 TCATGTGC 8660 8667 8 reverse hsa-miR-452 TCATCTGG 8221 8228 8 reverse hsa-miR-452 TCATGTGC 7945 7952 8 reverse hsa-miR-452 ACATCTGC 7508 7515 8 reverse hsa-miR-452 CCATCTGC 6787 6794 8 reverse hsa-miR-452 TCATCCGC 5912 5919 8 reverse hsa-miR-452 TCATCTGT 4053 4060 8 reverse hsa-miR-452 TCATCTCC 3667 3674 8 reverse hsa-miR-452 TCCTCTGC 3457 3464 8 reverse hsa-miR-452 TCTTCTGC 2210 2217 8 reverse hsa-miR-455-3p CAGTCCAT 13893 13900 8 reverse hsa-miR-455-5p TGTGTGCCTT 226 15641 15650 10 reverse hsa-miR-455-5p TCTGTGCCTT 227 11203 11212 10 reverse hsa-miR-455-5p TATGTGCTTT 228 10522 10531 10 reverse hsa-miR-483-3p CACTCCTC 13536 13543 8 reverse hsa-miR-483-3p CACTCCTC 10333 10340 8 reverse hsa-miR-483-3p CACTCCTC 6101 6108 8 reverse hsa-miR-486-5p TCATGTACT 9835 9843 9 reverse hsa-miR-486-5p TCCTGTCCT 6526 6534 9 reverse hsa-miR-487a AATCATACAG 229 12829 12838 10 reverse hsa-miR-487a AATCATACAG 229 933 942 10 reverse hsa-miR-491-5p AATGGGGAAG 230 14975 14984 10 reverse hsa-miR-491-5p AGAGGGGACC 231 12315 12324 10 reverse hsa-miR-491-5p AGTTGGGCAC 232 11555 11564 10 reverse hsa-miR-491-5p AGTAGAGAAC 233 6909 6918 10 reverse hsa-miR-491-5p GGTGAGGAAC 234 6005 6014 10 reverse hsa-miR-491-5p AGCGGGGCAC 235 4455 4464 10 reverse hsa-miR-491-5p AGTGGGAAAT 236 3846 3855 10 reverse hsa-miR-496 TTAGTATTA 10948 10956 9 reverse hsa-miR-496 TGAGTATAA 10768 10776 9 reverse hsa-miR-496 TCAGTATTA 9666 9674 9 reverse hsa-miR-501-3p ATGCATCAGG 237 15547 15556 10 reverse hsa-miR-501-3p ATCCACCGGG 238 11497 11506 10 reverse hsa-miR-501-3p AGGCACCAGG 239 2089 2098 10 reverse hsa-miR-504 AGACCCTGT 15325 15333 9 reverse hsa-miR-504 AGCCCCTGG 12898 12906 9 reverse hsa-miR-504 AGTCCCTGG 10591 10599 9 reverse hsa-miR-504 AGACCCGGG 4767 4775 9 reverse hsa-miR-508-3p TGATTATAGC 240 13565 13574 10 reverse hsa-miR-508-3p TGAGTGTAGC 241 3231 3240 10 reverse hsa-miR-512-3p CAGTGCTGTC 242 13211 13220 10 reverse hsa-miR-512-3p AAGTGCTCTC 243 7688 7697 10 reverse hsa-miR-512-3p AAGTGCTCTC 243 3184 3193 10 reverse hsa-miR-512-5p CACTCAG 14255 14261 7 reverse hsa-miR-512-5p CACTCAG 13591 13597 7 reverse hsa-miR-512-5p CACTCAG 12291 12297 7 reverse hsa-miR-512-5p CACTCAG 6652 6658 7 reverse hsa-miR-512-5p CACTCAG 5067 5073 7 reverse hsa-miR-514a-3p TTGACTCTT 14406 14414 9 reverse hsa-miR-514a-3p TTGACAGTT 13870 13878 9 reverse hsa-miR-514a-3p TTAACACTT 11237 11245 9 reverse hsa-miR-514a-3p ATGACACTT 10617 10625 9 reverse hsa-miR-515-3p GTGTGCCTT 15641 15649 9 reverse hsa-miR-515-3p GACTGCCTT 15539 15547 9 reverse hsa-miR-515-3p GAGTGACTT 1371 1379 9 reverse hsa-miR-516a-3p TGCTTCCT 10301 10308 8 reverse hsa-miR-517a-3p ATGGTGCATT 244 15650 15659 10 reverse hsa-miR-517a-3p ATCTTGCTTC 245 10303 10312 10 reverse hsa-miR-519b-3p AAAGTGCAT 13782 13790 9 reverse hsa-miR-519e-3p AAGTGCCTC 7048 7056 9 reverse hsa-miR-520a-5p CTCCAGATGG 6274 6283 10 reverse hsa-miR-545 CAGCAAGCACT 246 15050 15060 11 reverse hsa-miR-545 CAGAACACATT 247 11639 11649 11 reverse hsa-miR-545 CTGCAAACACT 248 3450 3460 11 reverse hsa-miR-549 TGACAACTGT 249 14327 14336 10 reverse hsa-miR-551b-3p GCTACCCAT 2411 2419 9 reverse hsa-miR-552 CACAGGTGA 15130 15138 9 reverse hsa-miR-552 AACAGGTCA 11407 11415 9 reverse hsa-miR-552 AACATGTGA 9513 9521 9 reverse hsa-miR-552 AACAGGTTA 2441 2449 9 reverse hsa-miR-552 AACAGGTAA 1569 1577 9 reverse hsa-miR-583 AAAAGAGGA 2921 2929 9 reverse hsa-miR-583 CAAATAGGA 2833 2841 9 reverse hsa-miR-583 CAACGAGGA 1824 1832 9 reverse hsa-miR-583 CAAAGAAGA 1139 1147 9 reverse hsa-miR-593-3p TGTCTCTGT 8204 8212 9 reverse hsa-miR-593-3p TGGCTCTGC 6852 6860 9 reverse hsa-miR-593-3p TGCCTCTGC 231 239 9 reverse hsa-miR-593-5p AGGCACCAG 2090 2098 9 reverse hsa-miR-593-5p AGGCACCAG 2083 2091 9 reverse hsa-miR-598 ACGTCATC 11432 11439 8 reverse hsa-miR-611 GCGAGGTCTC 250 4779 4788 10 reverse hsa-miR-611 GAGAGGCCCC 251 2121 2130 10 reverse hsa-miR-611 GAGAGGACCT 252 1546 1555 10 reverse hsa-miR-616-5p ACTCTAAAC 14510 14518 9 reverse hsa-miR-619 GACCTGGA 5824 5831 8 reverse hsa-miR-620 ATGAATATAG 253 14560 14569 10 reverse hsa-miR-620 ATGGAAATAT 254 12111 12120 10 reverse hsa-miR-620 TTGGATATAG 255 11026 11035 10 reverse hsa-miR-620 GTGGAGATGG 256 10397 10406 10 reverse hsa-miR-620 ATGGAGATCC 257 6268 6277 10 reverse hsa-miR-620 ATGGAGGGAG 258 5626 5635 10 reverse hsa-miR-620 CTGGAGAAAG 259 3827 3836 10 reverse hsa-miR-620 ATCCAGATAG 260 2959 2968 10 reverse hsa-miR-620 ATGGGGCTAG 261 2843 2852 10 reverse hsa-miR-620 AGGGAGAGAG 262 1551 1560 10 reverse hsa-miR-620 CAGGAGATAG 263 1430 1439 10 reverse hsa-miR-620 TTGGAGAGAG 264 1201 1210 10 reverse hsa-miR-623 TCCCTTGC 8306 8313 8 reverse hsa-miR-623 TCCCTTGC 5004 5011 8 reverse hsa-miR-631 CACCTGGCC 9900 9908 9 reverse hsa-miR-631 GACATGGCC 8632 8640 9 reverse hsa-miR-634 AACCAGCAC 4520 4528 9 reverse hsa-miR-636 TGTGCTTG 10386 10393 8 reverse hsa-miR-638 ACGGAGCGCG 265 4905 4914 10 reverse hsa-miR-638 AGGGAGGGCG 266 4615 4624 10 reverse hsa-miR-642a-5p ATCCCTCTC 8983 8991 9 reverse hsa-miR-642a-5p GTCCCTCCC 4722 4730 9 reverse hsa-miR-643 ACATGCATGC 267 15553 15562 10 reverse hsa-miR-643 CCTTGTAGGC 268 15378 15387 10 reverse hsa-miR-643 TCTTGTATTC 269 14423 14432 10 reverse hsa-miR-643 ACTGGTATGT 270 13933 13942 10 reverse hsa-miR-643 ACTTCTATTC 271 12886 12895 10 reverse hsa-miR-643 ACTTTTCTGC 272 12044 12053 10 reverse hsa-miR-643 GCTTGTAAGC 273 11698 11707 10 reverse hsa-miR-643 AGTTGTATGT 274 10531 10540 10 reverse hsa-miR-643 ACTTGGAAGC 275 8105 8114 10 reverse hsa-miR-643 ACTTGTGTGG 276 7227 7236 10 reverse hsa-miR-643 ACTTGTTTGA 277 1880 1889 10 reverse hsa-miR-643 ACATGTTTGC 278 1695 1704 10 reverse hsa-miR-650 AGGAGGCAC 279 9647 9655 9 reverse hsa-miR-650 AGAAGGCAG 280 6917 6925 9 reverse hsa-miR-650 AGGAGCCAG 281 3474 3482 9 reverse hsa-miR-650 ATGAGGCAG 282 3052 3060 9 reverse hsa-miR-651 TCATGATAAG 283 15700 15709 10 reverse hsa-miR-651 TTAGGTTAAA 284 13993 14002 10 reverse hsa-miR-651 TTAAAATAAG 285 13988 13997 10 reverse hsa-miR-651 TTAGCATAAC 286 12788 12797 10 reverse hsa-miR-651 TTATGATGAG 287 12617 12626 10 reverse hsa-miR-651 TTTGGATGAG 288 11069 11078 10 reverse hsa-miR-651 TGAGTATAAG 289 10767 10776 10 reverse hsa-miR-651 TTACAATAAG 290 10546 10555 10 reverse hsa-miR-651 TAAGGATAAA 291 8265 8274 10 reverse hsa-miR-651 TGTGGATAAG 292 7222 7231 10 reverse hsa-miR-651 GTAGGATAGG 293 5553 5562 10 reverse hsa-miR-651 CTAGGAAAAG 294 2823 2832 10 reverse hsa-miR-651 CTATGATAAG 295 1635 1644 10 reverse hsa-miR-651 TAAGGATAGG 296 1562 1571 10 reverse hsa-miR-654-3p TATGTATACT 297 15493 15502 10 reverse hsa-miR-654-3p TATCTCTTCT 298 14775 14784 10 reverse hsa-miR-654-3p TCTATCTGCT 299 8354 8363 10 reverse hsa-miR-654-3p AATGTCTGGT 300 6720 6729 10 reverse hsa-miR-654-3p TATGTTTCCT 301 6638 6647 10 reverse hsa-miR-654-3p TTTTTCTGCT 302 6586 6595 10 reverse hsa-miR-654-3p TATGTCTTTT 303 6534 6543 10 reverse hsa-miR-654-3p TATATCTGCA 304 6214 6223 10 reverse hsa-miR-654-3p TATGTAGGCT 305 97 106 10 reverse hsa-miR-655 GTAATACAT 15593 15601 9 reverse hsa-miR-655 ATAGTACAT 4200 4208 9 reverse hsa-miR-655 ATAAGACAT 3642 3650 9 reverse hsa-miR-655 ATAATACAG 2265 2273 9 reverse hsa-miR-655 ACAATACAT 1757 1765 9 reverse hsa-miR-656 AATATTATA 657 665 9 reverse hsa-miR-664-3p TATTCATTT 9385 9393 9 reverse hsa-miR-765 TGGAGGA 5020 5026 7 reverse hsa-miR-766 CTCCAGCCCC 306 12901 12910 10 reverse hsa-miR-766 CTCCAGCCCC 307 5032 5041 10 reverse hsa-miR-767-3p CCTGCTCAT 14871 14879 9 reverse hsa-miR-767-3p TCTTCTCAT 9155 9163 9 reverse hsa-miR-875 CCTGGAAATA 308 5820 5829 10 reverse hsa-miR-875 CCTAGAAACA 309 5294 5303 10 reverse hsa-miR-876 TGGATTTCT 6366 6374 9 reverse hsa-miR-876 TGGATTTCT 142 150 9 reverse hsa-miR-888-3p GACTGACTCC 310 15772 15781 10 reverse hsa-miR-888-3p GACTGACAGC 311 9119 9128 10 reverse hsa-miR-890 TACTTGGAAG 312 8106 8115 10 reverse hsa-miR-940 AAGGCAGTG 1807 1815 9 reverse hsa-miR-941 CACCCAGGT 14396 14404 9 reverse hsa-miR-941 CACCCTGCC 13715 13723 9 reverse hsa-miR-941 CACCCCTCT 13128 13136 9 reverse hsa-miR-941 CACTCAGCT 12289 12297 9 reverse hsa-miR-941 CTCCCGGGT 10102 10110 9 reverse hsa-miR-941 CAGCCTGCT 10034 10042 9 reverse hsa-miR-941 CACCCACCT 9904 9912 9 reverse hsa-miR-941 CACCTGGCC 9900 9908 9 reverse hsa-miR-941 CATCTGGCT 8219 8227 9 reverse hsa-miR-941 CACTCGACT 8148 8156 9 reverse hsa-miR-941 CTCCCAGCT 6840 6848 9 reverse hsa-miR-941 CTCACGGCT 6031 6039 9 reverse hsa-miR-941 CAGCCCGCT 5928 5936 9 reverse hsa-miR-941 CACCTGACT 5510 5518 9 reverse hsa-miR-941 CACGCCGCT 5142 5150 9 reverse hsa-miR-941 CTCCCTGCT 3983 3991 9 reverse hsa-miR-941 CACCAGGCA 2087 2095 9 reverse hsa-miR-941 CTCCCGGGT 390 398 9 reverse hsa-miR-941 CACCCAGCC 186 194 9 reverse hsa-miR-941-2 ATCCGACTGT 313 9657 9666 10 reverse hsa-miR-941-2 TCCCTGCTGT 314 8726 8735 10 reverse hsa-miR-941-2 TCCCAGCTGT 315 6838 6847 10 reverse hsa-miR-941-2 AGCCCGCTGT 316 5926 5935 10 reverse hsa-miR-941-2 ACCCGGGCGT 317 4764 4773 10 reverse hsa-miR-1179 AAGTATCCTTT 318 15346 15356 11 reverse hsa-miR-1179 ATGCATTCTGT 319 3357 3367 11 reverse hsa-miR-1179 ATGCATTCTCT 320 1854 1864 11 reverse hsa-miR-1207-5p TGGCAGGG 11441 11448 8 reverse hsa-miR-1224-3p CTCCACCTCC 321 399 408 10 reverse hsa-miR-1228-3p TCCCACCTG 13637 13645 9 reverse hsa-miR-1228-3p TCACGCCTG 4992 5000 9 reverse hsa-miR-1231 GTGTCTGGC 12807 12815 9 reverse hsa-miR-1231 GTGTCCGGG 4739 4747 9 reverse hsa-miR-1245 AAGTGATCT 8341 8349 9 reverse hsa-miR-1245 AAGTGATCT 2020 2028 9 reverse hsa-miR-1249 CGCCCTTC 5907 5914 8 reverse hsa-miR-1251 ACTCTAGGT 12854 12862 9 reverse hsa-miR-1251 ACTCTATCT 8357 8365 9 reverse hsa-miR-1251 ACTCCAGCT 4044 4052 9 reverse hsa-miR-1251 AGTCTAGCT 457 465 9 reverse hsa-miR-1252 AGAGGGAAAT 322 3819 3828 10 reverse hsa-miR-1252 GGAAGGAAAT 323 1625 1634 10 reverse hsa-miR-1268 CGGGCGTGG 4762 4770 9 reverse hsa-miR-1270 CTGGAAATA 5820 5828 9 reverse hsa-miR-1270 CTGGAGATG 5055 5063 9 reverse hsa-miR-1270 CTGGAGAAA 3828 3836 9 reverse hsa-miR-1270 CAGGAGATA 1431 1439 9 reverse hsa-miR-1272 GATGATGA 10622 10629 8 reverse hsa-miR-1275 GTAGGGGAGA 324 1189 1198 10 reverse hsa-miR-1302 ATGGGACACA 325 15021 15030 10 reverse hsa-miR-1302 TTTGGATATA 326 11027 11036 10 reverse hsa-miR-1302 TTAGGGCATA 327 8421 8430 10 reverse hsa-miR-1302 TTGGAACAGA 328 6076 6085 10 reverse hsa-miR-1302 CTGGGACTTA 329 4819 4828 10 reverse hsa-miR-1302 GTGGGAAATA 330 3845 3854 10 reverse hsa-miR-1302 TTGTGAGATA 331 1944 1953 10 reverse hsa-miR-1302 CTGGGAAATA 332 867 876 10 reverse hsa-miR-1324 TCAAGACAGA 333 9426 9435 10 reverse hsa-miR-1827 TGAGGCAGT 3051 3059 9 reverse hsa-miR-1911-3p CACCAGGCA 2087 2095 9 reverse hsa-miR-1915 CCCCAGGG 5111 5118 8 reverse hsa-miR-2909 TTTAGGGCC 3728 3736 9 reverse

B. Another exemplary multiple miRNAs-one mRNA paradigm involves UCP2.

UCP2 is a mitochondrial transporter protein expressed in WAT, skeletal muscle, pancreatic islets and the central nervous system. Like UCP1, it creates proton leaks across the inner mitochondrial membrane, thus uncoupling oxidative phosphorylation from ATP synthesis (adaptive thermogenesis, see FIG. 5) (Lowell et al., Nature (2000)).

Two recent meta-analyses report an association between polymorphisms in the promoter region of UCP2 and obesity (Liu et al., Gene (2013); Andersen et al., Int J. Obes. (2013)). The first meta-analysis included 14 studies (7,647 cases and 11,322 controls) and concluded that there is a significant association of the A allele of the UCP2-866G/A polymorphism with reduced risk of obesity, especially in European populations. In the second meta-analysis including 12,984 subjects, the common UCP2-866G allele is associated with obesity. The same UCP2-866G allele is associated with decreased insulin sensitivity in 17,636 Danish subjects. In a study, UCP2 mRNA levels in visceral fat were decreased in subjects with the GG phenotype (Esterbauer et al., Nat. Genet. (2001)). A trend toward a negative correlation between subcutaneous adipocyte UCP2 mRNA and percent body fat was found in another study (Wang et al., American Journal of Physiol. (2004)). This information supports targeting UCP2 expression and activity as a meaningful way to alter adaptive thermogenesis and consequently treat human obesity. Many strategies could be implemented to achieve this goal, however, the one employed in the methods of the invention uses miRNA agents to modulate simultaneously several elements within the thermogenic pathways to increase UCP2 synthesis and activity. Both direct and indirect interactions between miRNAs and the UCP2 gene are considered. Direct interaction means the direct binding of miRNAs to the various regions of the UCP2 gene, resulting in alterations of the transcription, translation, stability and/or degradation of the UCP1 mRNA. Indirect interaction means that miRNAs alter the transcription, translation, stability and/or degradation of thermogenic mRNAs, whose expressed proteins alter the transcription of the UCP2 gene. Furthermore, indirect interaction means that miRNAs alter the transcription, translation, stability and/or degradation of other miRNAs that modify the transcription of the UCP2 gene.

The promoter region of the human UCP2 gene (ENSG00000175567, Homo sapiens uncoupling protein 2 (mitochondrial, proton carrier) (UCP2), RefSeqGene on chromosome 11) is rich is regulatory element motifs:

UCP2 Gene Regulatory Elements: 1. RXR/T3RE Motif: AGGTCA Eight Length: 6, Interval: 1,074->1,079; 3,083->3,088; 3,239->3,244; 4,304->4,309; 6,965->6,970; 7,420->7,425; 7,677->7,682; 13,319->13,324; Mismatches: 0. 2. GC Box 1 Motif: CGCCC Sixteen

Length: 5, Interval: 2,605->2,609; 4,323->4,327; 4,523->4,527; 4,933->4,937; 4,959->4,963; 5,048->5,052; 5,066->5,070; 5,146->5,150; 5,155->5,159; 5,387->5,391; 5,483->5,487; 6,067->6,071; 8,523->8,527; 9,790->9,794; 10,819->10,823; 11,754->11,758; Mismatches: 0.

3. GC Box 2 Motif: GCGGG Five Length: 5, Interval: 4,263->4,267; 4,757->4,761; 4,860->4,864; 7,619->7,623; 11,262->11,266; Mismatches: 0. 4. GT Box 1 Motif: CACCC Thirty

Length: 5, Interval: 1,421->1,425; 1,677->1,681; 1,761->1,765; 1,825->1,829; 1,833->1,837; 2,036->2,040; 3,003->3,007; 4,903->4,907; 4,947->4,951; 5,210->5,214; 6,204->6,208; 6,247->6,251; 6,469->6,473; 6,828->6,832; 7,681->7,685; 8,048->8,052; 8,437->8,441; 8,572->8,576; 8,599->8,603; 8,702->8,706; 11,077->11,081; 11,235->11,239; 12,006->12,010; 12,374->12,378; 13,475->13,479; 13,666->13,670; 13,687->13,691; 13,838->13,842; 14,410->14,414; 14,545->14,549; Mismatches: 0.

5. GT Box 2 Motif: GTGGG Twenty Six

Length: 5, Interval: 123->127; 1,006->1,010; 2,105->2,109; 4,562->4,566; 5,793->5,797; 6,029->6,033; 6,034->6,038; 6,040->6,044; 6,150->6,154; 7,271->7,275; 7,392->7,396; 9,040->9,044; 9,697->9,701; 10,227->10,231; 10,238->10,242; 10,247->10,251; 11,817->11821; 12,410->12,414; 12,414->12,418; 12,678->12,682; 13,047->13,051; 13,238->13,742; 13,743->13,747; 14,252->14,256; 14,969->14,973; 15,104->15,108; Mismatches: 0.

6. CpG Methylation Island Motif: CG

Two Hundred and Ninety Five, including many between positions 4,071 to 5,212.

FIG. 8B depicts the location of these various regulatory elements in reference to the UCP2 transcription start site at nucleotide position 5,001 of the 15,174 base pair human UCP2 gene. Direct or indirect activation or repression of these regulatory elements by miRNAs will result in alterations of UCP2 gene expression and activity.

A survey of miRNAs targeting the human UCP2 3′UTR with several prediction programs, using the UCP2 Ensembl 2,113 base pair transcript ENST00000310473 as a target revealed binding sites for 161 miRNAs as shown in Table 9.

TABLE 9 miRNAs with predicted binding sites in the 3′UTR of UCP2 transcript sequence. hsa-miR-1 hsa-miR-1-2 hsa-miR-101-1 hsa-miR-101-2 hsa-miR-103 hsa-miR-105-1 hsa-miR-105-2 hsa-miR-106b hsa-miR-107 hsa-miR-1204 hsa-miR-1207 hsa-miR-1208 hsa-miR-1226 hsa-miR-1246 hsa-miR-1252 hsa-miR-1253 hsa-miR-1255a hsa-miR-1255b-1 hsa-miR-1255b-2 hsa-miR-1260a hsa-miR-1262 hsa-miR-1263 hsa-miR-1265 hsa-miR-1275 hsa-miR-1276 hsa-miR-1278 hsa-miR-1285-1 hsa-miR-1286 hsa-miR-1293 hsa-miR-1300 hsa-miR-1302-1 hsa-miR-1302-10 hsa-miR-1302-11 hsa-miR-1302-2 hsa-miR-1302-3 hsa-miR-1302-4 hsa-miR-1302-5 hsa-miR-1302-6 hsa-miR-1302-7 hsa-miR-1302-8 hsa-miR-1302-9 hsa-miR-1303 hsa-miR-130a hsa-miR-1321 hsa-miR-138-1 hsa-miR-138-2 hsa-miR-149 hsa-miR-150-3p hsa-miR-150-5p hsa-miR-1538 hsa-miR-155 hsa-miR-15a hsa-miR-15b hsa-miR-16-1 hsa-miR-16-2 hsa-miR-184 hsa-miR-185-3p hsa-miR-185-5p hsa-miR-186 hsa-miR-188 hsa-miR-18a hsa-miR-18b hsa-miR-193a hsa-miR-195 hsa-miR-199b hsa-miR-200a hsa-miR-203 hsa-miR-206 hsa-miR-214 hsa-miR-219-1 hsa-miR-219-2 hsa-miR-221-5p hsa-miR-23b hsa-miR-24-1 hsa-miR-24-2 hsa-miR-27b-5p hsa-miR-28 hsa-miR-296-3p hsa-miR-296-5p hsa-miR-3064 hsa-miR-323a hsa-miR-328 hsa-miR-330 hsa-miR-331 hsa-miR-338 hsa-miR-342 hsa-miR-3619 hsa-miR-370 hsa-miR-377 hsa-miR-378a hsa-miR-383 hsa-miR-411 hsa-miR-412 hsa-miR-422a hsa-miR-424 hsa-miR-425 hsa-miR-4291 hsa-miR-432-3p hsa-miR-4505 hsa-miR-450b hsa-miR-453 hsa-miR-4533 hsa-miR-4539 hsa-miR-4745 hsa-miR-4747 hsa-miR-485-5p hsa-miR-486 hsa-miR-490 hsa-miR-491 hsa-miR-493 hsa-miR-497 hsa-miR-498 hsa-miR-503 hsa-miR-505 hsa-miR-508-3p hsa-miR-532 hsa-miR-539 hsa-miR-541 hsa-miR-5481 hsa-miR-552 hsa-miR-563 hsa-miR-575 hsa-miR-577 hsa-miR-580 hsa-miR-583 hsa-miR-584 hsa-miR-608 hsa-miR-612 hsa-miR-613 hsa-miR-615-3p hsa-miR-618 hsa-miR-625 hsa-miR-626 hsa-miR-634 hsa-miR-635 hsa-miR-638 hsa-miR-645 hsa-miR-646 hsa-miR-647 hsa-miR-652 hsa-miR-654 hsa-miR-658 hsa-miR-663a hsa-miR-663b hsa-miR-664-5p hsa-miR-675 hsa-miR-7-1 hsa-miR-7-2 hsa-miR-7-3 hsa-miR-708 hsa-miR-761 hsa-miR-765 hsa-miR-769 hsa-miR-770 hsa-miR-876 hsa-miR-877 hsa-miR-921 hsa-miR-922 hsa-miR-92a-1 hsa-miR-92a-2-5p hsa-miR-92b

Moreover, a survey of miRNAs targeting the human UCP2 5′UTR with several prediction programs, using the human UCP2 gene (ENSG00000175567, 15,174 base pair (bp) of the, including 5,000 bp 5′UTR as a target revealed binding sites for 54 miRNAs in UCP2 5′UTR as shown in Table 10.

TABLE 10 miRNAs with predicted binding sites in the 5′UTR of UCP2 gene sequence. Seed SEQ ID MicroRNA Length Start Sequence NO End P value hsa-let-7c 9 3052 UAGAGUUAC 3044 0.0374 hsa-let-7i-3p 9 3051 CUGCGCAAG 3043 0.0374 hsa-miR-1228-5p 9 3419 UGGGCGGGG 3411 0.0374 hsa-miR-1229-3p 9 3419 UCUCACCAC 3411 0.0374 hsa-miR-129-1-3p 10 2784 AGCCCUUACC 334 2775 0.0095 hsa-miR-1302 9 4219 UGGGACAUA 4211 0.0374 hsa-miR-1303 9 2159 UUAGAGACG 2151 0.0374 hsa-miR-136 9 4486 CUCCAUUUG 4478 0.0374 hsa-miR-155 9 2160 UUAAUGCUA 2152 0.0374 hsa-miR-16 10 3603 UAGCAGCACG 335 3594 0.0095 hsa-miR-18a-3p 10 3603 ACUGCCCUAA 336 3594 0.0095 hsa-miR-190 9 2428 UGAUAUGUU 2420 0.0374 hsa-miR-191 9 3052 CAACGGAAU 3044 0.0374 hsa-miR-192 9 4390 CUGACCUAU 4382 0.0374 hsa-miR-194 9 1643 UGUAACAGC 1635 0.0374 hsa-miR-197 9 5001 UCACCACCU 4993 0.0374 hsa-miR-19b-2-5p 10 3052 AGUUUUGCAG 337 3043 0.0095 hsa-miR-203 9 3051 UGAAAUGUU 3043 0.0374 hsa-miR-218 10 3603 UUGUGCUUGA 338 3594 0.0095 hsa-miR-218-1-3p 9 5001 UGGUUCCGU 4993 0.0374 hsa-miR-219-1-3p 9 3614 AGAGUUGAG 3606 0.0374 hsa-miR-26a-2-3p 9 2163 CCUAUUCUU 2155 0.0374 hsa-miR-27a-3p 10 3603 UUCACAGUGG 339 3594 0.0095 hsa-miR-27a-5p 11 3336 AGGGCUUAGCU 340 3326 0.0024 hsa-miR-28-5p 10 3603 AAGGAGCUCA 341 3594 0.0095 hsa-miR-331-3p 9 4134 GCCCCUGGG 4126 0.0374 hsa-miR-337-5p 10 115 GAACGGCUUC 342 106 0.0095 hsa-miR-340-3p 9 1872 CCGUCUCAG 1864 0.0374 hsa-miR-34c-3p 11 2162 AAUCACUAACC 343 2152 0.0024 hsa-miR-373-5p 11 530 ACUCAAAAUGG 344 520 0.0024 hsa-miR-425 9 1013 AAUGACACG 1005 0.0374 hsa-miR-497 9 3661 AGCAGCACA 3653 0.0374 hsa-miR-501-5p 9 4164 AUCCUUUGU 4156 0.0374 hsa-miR-505 9 1015 GUCAACACU 1007 0.0374 hsa-miR-508-3p 9 1274 GAUUGUAGC 1266 0.0374 hsa-miR-509-3p 12 2554 UGAUUGGUACGU 345 2543 0.0006 hsa-miR-512-5p 10 987 ACUCAGCCUU 346 978 0.0095 hsa-miR-514 9 5001 UUGACACUU 4993 0.0374 hsa-miR-515-5p 9 59 UUCUCCAAA 51 0.0374 hsa-miR-518a-3p 9 19 GAAAGCGCU 11 0.0374 hsa-miR-519e-5p 11 2525 UCUCCAAAAGG 347 2515 0.0024 hsa-miR-548a-3p 10 680 CAAAACUGGC 348 671 0.0095 hsa-miR-550a-3p 9 4312 GUCUUACUC 4304 0.0374 hsa-miR-571 9 739 UGAGUUGGC 731 0.0374 hsa-miR-578 9 1377 CUUCUUGUG 1369 0.0374 hsa-miR-606 9 4420 AACUACUGA 4412 0.0374 hsa-miR-615-5p 10 1140 GGGGGUCCCC 349 1131 0.0095 hsa-miR-638 9 2710 GGGAUCGCG 2702 0.0374 hsa-miR-657 12 1316 GCAGGUUCUCAC 350 1305 0.0006 hsa-miR-658 9 3673 GGCGGAGGG 3665 0.0374 hsa-miR-877-3p 9 4349 UCCUCUUCU 4341 0.0374 hsa-miR-93-3p 9 799 ACUGCUGAG 791 0.0374 hsa-miR-96-3p 9 799 AAUCAUGUG 791 0.0374 hsa-miR-99b-3p 9 2163 CAAGCUCGU 2155 0.0374 c) A Multiple microRNAs-Multiple mRNAs Paradigm.

The 83 thermogenic regulator molecules selected in Table 1 were screened for high stringency Multiple miRNAs-Multiple mRNAs associations. The results of these analyses with 7 major prediction tools are shown in FIG. 4. The union of these 7 tools produces 4439 miRNA-gene couples. Overlap between these tools decreases as the number of tools increases, reaching only 15 miRNA-gene couples when 7 tools are considered.

d) An Over-Representation of One microRNA Seed Sequence Motif Among Co-Regulated mRNA Targets Paradigm.

Several approaches can be used to identify pathway-specific miRNAs. For example, searching the 3′-UTRs of putatively co-regulated genes for an over-represented sequence from a miRNA seed region could identify a common regulatory miRNA. To determine if particular miRNA seed sequences were overrepresented among the 3′ UTR of the chosen 83 thermogenesis targets, the miRvestigator web application (mirvestigator.systemsbiology.net/) was employed. Using the following parameters (motif size of 8 bp, default Weeder model, seed model of 8 mer, 100% complementarity homology and 0.25 wobble base-pairing allowed), it was determined that that the motif 5′-UUUGUACA-3′ recognized by hsa-miR-19a/-19b is over represented among 15 of the 83 thermogenesis targets with a complementarity p value of 1.7×10⁻⁰⁴ as shown in Table 11. Of note is that hsa-miR-19 has been reported as an abundant adipocyte miRNA.

TABLE 11 Complimentarity between the common motif UUUGUACA and hsa-miR-19a/19b. Seed Length of Complementary Complimentarity miRNA Name miRNA Seed Model Complimentarity Base-Pairing P-Value hsa-miR-19a UGUGCAAA 8 mer 8 Motif 5′ UUUGUACA 3′ 1.7e-04 hsa-miR-19b          ||||:|||       3′ AAACGUGU 5′ miRNA Seed

The Minimum Free Energy levels of the hsa-miR-19 mRNA/miRNA duplexes identified by miRvestigator were quite low, favoring tight binding. Accordingly, the miRvestigator analysis was repeated with less stringent levels of complementarity. This analysis identified a further 10 additional targets (CEBPD, PRKAA1, TWIST1, IRS1, NCOA1, NCOA2, NCOA3, KLF5, RPS6KB1, NRIP1) with 95% similarity to the consensus hsa-miR-19 motif. Interestingly, hsa-miR-19 is among the most abundant miRNAs in adipose tissue. The genes identified as containing a sequence complementary to hsa-miR-19 seed region are set forth in Table 12.

TABLE 12 Thermogenic regulators identified as targets for hsa-miR-19. Start  % Similarity Relative  to Consensus Minimum Free Gene Sequence to Stop Motif (Quality = Energy (MFE) of Gene symbol of site Codon (bp) High|Medium|Fair) mRNA-miRNA Duplex 650 BMP2 UUUGUACA  386 100.00 -6.80 1052 CEBPD UUUGUAAA  263  95.44 -3.40 7132 TNFRSF1A UUUGUACA  510 100.00 -6.80 5562 PRKAA1 UUUGUAAA 2400  95.44 -3.40 5563 PRKAA2 UUUGUACA  542 100.00 -6.80 655 BMP7 UUUGUACA 1927 100.00 -6.80 652 BMP4 UUUGUACA  770 100.00 -6.80 133522 PPARGC1B UUUGUACA 7199 100.00 -6.80 7474 WNT6A UUUGUACA 1414 100.00 -6.80 6720 SREBF1 UUUGUACA  510 100.00 -6.80 7281 TWIST1 UUUGUAAA  649  95.44 -3.40 3667 IRS1 UUUGUAAA  992  95.44 -3.40 10499 NCOA2 UUUGUAAA 1381  95.44 -3.40 8204 NRIP1 UUUGUACA 1718 100.00 -6.80 8204 NRIP1 UUUGUAAA 1935  95.44 -3.40 8202 NCOA3 UUUGUAAA  965  95.44 -3.40 1385 CREB1 UUUGUAAA 1973  95.44 -3.40 1385 CREB1 UUUGUACA 2822 100.00 -6.80 1385 CREB1 UUUGUACA 2822 100.00 -6.80 1385 CREB1 UUUGUAAA 4175  95.44 -3.40 3643 INSR UUUGUAAA 2105  95.44 -3.40 8013 NR4A3 UUUGUACA 2347 100.00 -6.80 860 RUNX2 UUUGUACA 2425 100.00 -6.80 6776 STAT5A UUUGUACA 1214 100.00 -6.80 1874 E2F4 UUUGUACA  755 100.00 -6.80 688 KLF5 UUUGUAAA  549  95.44 -3.40 8648 NCOA1 UUUGUAAA  381  95.44 -3.40 6198 RPS6KB1 UUUGUAAA 2531  95.44 -3.40

Accordingly, the miRvestigator analysis was repeated with less stringent levels of complementarity (motif size of 8 bp, default Weeder model, seed model of 8 mer, 95% complementarity homology and 0.25 wobble base-pairing allowed). This analysis identified a further 10-12 additional targets (CEBPD, CREB1, PRKAA1, TWIST1, INSR, IRS1, NCOA1, NCOA2, NCOA3, KLF5, RPS6KB1, NRIP1) with 95% similarity to the consensus hsa-miR-19 motif. Interestingly, hsa-miR-19 is among the most abundant miRNAs in adipose tissue. The genes identified as containing a sequence complementary to hsa-miR-19 seed region are set forth in Table 13.

TABLE 13 Thermogenic regulators identified as targets for hsa-miR-19a/b with 95% to 100% similarity to consensus motif Start  % Similarity Relative  to Consensus Minimum Free Gene Sequence to Stop Motif (Quality = Energy (MFE) of Gene symbol of site Codon (bp) High|Medium|Fair) mRNA-miRNA Duplex 650 BMP2 UUUGUACA  386 100.00 -6.80 1052 CEBPD UUUGUAAA  263  95.42 -3.40 7132 TNFRSF1A UUUGUACA  510 100.00 -6.80 4040 LRP8 UUUGUACA  151 100.00 -6.80 4040 LRP8 UUUGUAAA 4965  95.42 -3.40 5562 PRKAA1 UUUGUAAA 2400  95.42 -3.40 5563 PRKAA2 UUUGUACA  542 100.00 -6.80 655 BMP7 UUUGUACA 1927 100.00 -6.80 652 BMP4 UUUGUACA  770 100.00 -6.80 133522 PPARGC1B UUUGUACA 7199 100.00 -6.80 1874 E2F4 UUUGUACA  755 100.00 -6.80 7474 WNT5A UUUGUACA 1414 100.00 -6.80 8720 SREBF1 UUUGUACA  510 100.00 -6.80 7291 TWIST1 UUUGUAAA  649  95.42 -3.40 3667 IRS1 UUUGUAAA  992  95.42 -3.40 10499 NCOA2 UUUGUAAA 1381  95.42 -3.40 8204 NRIP1 UUUGUACA 1718 100.00 -6.80 8204 NRIP1 UUUGUAAA 1935  95.42 -3.40 8202 NCOA3 UUUGUAAA  965  95.42 -3.40 3643 INSR UUUGUAAA 2105  95.42 -3.40 8013 NR4A3 UUUGUACA 2347 100.00 -6.80 6776 STAT5A UUUGUACA 1214 100.00 -6.80 688 KLF5 UUUGUAAA  548  95.42 -3.40 8648 NCOA1 UUUGUAAA  381  95.42 -3.40 860 RUNX2 UUUGUACA 2425 100.00 -6.80 1385 CREB1 UUUGUAAA 1973  95.42 -3.40 1385 CREN1 UUUGUACA 2822 100.00 -6.80 1385 CREB1 UUUGUAAA 4175  95.42 -3.40 6198 PRS6KB1 UUUGUAAA 2531  95.42 -3.40 Without wobbling, the same motif 5′-UUUGUACA-3′ is overrepresented among targets of hsa-miR-1283 with a complementarity p value of 1.4×10⁻⁴. Furthermore, hsa-miR-1283 binds to other mRNAs of interest like ABCA1 (cholesterol transporter), the adiponectin receptor and the transcription factor TCF7L2 that is implicated in genetic human obesity.

Similarly, other miRNA over-represented seed sequences were identified for miRNAs expressed in adipocytes. They include the universal hsa-let-7 family (sequence CUAUACAA, p value=7.5e-04) and the adipocyte-rich hsa-miR-30 family (sequence UGUAAACA, p value=1.9×10⁻³) to name a few.

With respect to PRDM16, CIDEA, NRIP1, KDM3A, CEPPB, PPARG, PPARGC1A, and PPKAA2, which according to the STRING software package are directly linked to UCP1, it appears that all of them share (at motif size 8 bp, default weeder model, seed model 8 mer, 95% complementarity homology and 0.25 wobble base-pairing allowed) a consensus sequence with several miRNAs, including hsa-miR-3658 (p value=1.9e-003) and the hsa-miR-30 family (p value=6.3e-003) as follows:

hsa-miR-3658: Motif 5′ UUUUUUAC- 3′           ||:||||       3' -AAGAAUUU 5' miRNA Seed hsa-miR-30a/b/c/d/e: Motif 5′ UUUUUUAC- 3′          ||||||||       3' -AAGAAUUU 5' miRNA Seed e) An Intronic miRNA-Multiple mRNAs Pathway-Specific Paradigm.

Many mammalian miRNAs are located within introns of protein-coding genes rather than in their own unique transcription units. Intronic miRNAs are typically expressed and processed with the precursor mRNA in which they reside. Although the intronic miRNAs and their host genes can be regulated independently, an intronic miRNA can down-regulate its own host protein-coding gene by targeting the host gene's UTR. Feedback regulation on host protein-coding genes could be achieved by selecting the transcription factors that are miRNA targets or by protein-protein interactions between intronic miRNA host gene product and miRNA target gene products. As an example, miR-33 acts in concert with the SREBP host genes to control cholesterol homeostasis and the pharmacological inhibition of miR-33a and miR-33b is a promising therapeutic strategy to raise plasma HDL and lower VLDL triglyceride levels for the treatment of dyslipidaemias.

Examination of the 83 thermogenic target genes reveals two intronic miRNAs: miR-378 located in the PPARGC1B gene and miR-4251 located in the PRDM16 gene.

Mining of the Internet tools predicting miRNA targets indicates that miR-378 targets include BMP2, PPARA, PPARGC1A, PRDM16, STATS and WNT10A as well as ADIPOQ and IGFR1; and that miR-4251 targets include BMP2, CTBP1, CTBP2, MAPK14, NCOA3, PLACE, PPARA, PPARD, TRPM8, as well as ABCA5, ABCA13, ADIPOQR2, KDM5B, KLF-12, KLF-14 and TCF7L2.

Example 3 High-Content Cellular Phenotypic Screening

High-content screening methods are used to screen for novel miRNA agents that modulate the activity of thermogenic regulators (e.g., UCP1 and UCP2). High-content screening is a drug discovery method that uses images of living cells to facilitate molecule discovery. Such automated image based screening methods are particularly suitable for identifying agents which alter cellular phenotypes.

WAT cells which contain a single large lipid droplet, whereas, in contrast, BAT cells contain numerous smaller droplets and a much higher number of mitochondria, which contain iron and make them appear brown. The large number of mitochondria in BAT leads to an increased oxygen consumption, when compared to WAT. Accordingly, it is possible to distinguish between BAT and WAT cells visually based on their cellular phenotype.

Accordingly, high-content screening methods were used to screen for novel miRNA agents that modulate the activity of thermogenic regulators. Specifically, the phenotypic appearance of cultured human adipocytes and adipose tissue derived mesenchymal stem cells grown in the presence and absence of miRNA agonists or antagonists was assessed over two weeks by phase contrast microscopy of the cultured cells, measurement of the cellular lipid content (using Oil Red 0 Staining or Nile Red fluorescence); mitochondrial content (e.g., using Life Technologies Mito-Tracker Red FM), and/or oxygen consumption in vitro (e.g., using the Seahorse Bioscience Extra-Cellular Flux Instrument). mRNA expression is measured by targeted q-RT-PCR and universal RNA-Sequencing. Protein expression is measured by targeted Western Blotting and universal proteomic profiling.

A. Differentiation of Human Pre-Adipocytes into Adipocytes.

1. Differentiation Protocol.

In order to assess the effect of miRNA analogs on human pre-adipocytes differentiation into mature adipocytes, human subcutaneous pre-adipocytes (SuperLot 0048 from 8 female donors, ZenBio, NC) were plated on Day 0 into 96-well plates and allowed to attach overnight in preadipocyte medium (DMEM/Ham's F-12 (1:1, v/v), HEPES buffer, Fetal bovine serum and Antibiotics). The next day (Day 1), the medium was removed and replaced with differentiation medium (DMEM/Ham's F-12 (1:1, v/v), 100 μM Ascorbic Acid, 0.85 μM insulin, 20 nM sodium selenite, 0.2 nM, triiodothryonine, 1 μM dexamethasone, 100 μM isobutyl-methylxanthine, 100 nM Rosiglitazone and Antibiotics. The cells were allowed to incubate for 2 days at 37°, 5% CO₂. After 2 days (Day 3), the medium was removed and replaced with fresh maintenance medium (DMEM/Ham's F-12 (1:1, v/v), 100 μM Ascorbic Acid, 0.85 μM insulin, 20 nM sodium selenite, 0.2 nM triiodothryonine, and Antibiotics). On Day 3, the cells were transfected with miRNA analogs (Dharmacon specific miRIDIAN Mimics and Hairpin Inhibitors) using the transfecting agent Dharmafect1. All treatments were in triplicate. Post transfection, the negative control was maintenance medium only and the positive control was maintenance medium with 100 nM of the PPARG agonist rosiglitazone. After 2 days, medium was removed and replaced with fresh maintenance medium. The maintenance medium then changed every two days until the end of the treatment period (Day 15). At the end of the treatment (total of 15 days in culture) cells were processed for Phenotyping and Genotyping Screening.

1. Transfection of Pre-Adipocytes.

Transfection reagents are used to facilitate the penetration of miRNA analogs into target cells. As an example, the extent of transfection efficiency we achieved in pre-adipocytes with the transfecting agent Dharmafect 1 (Dharmacon, CO) is depicted herein. Transfection efficiency was assessed in two ways:

a. Measurement of Cellular Epifluorescence after Transfection with Fluorescent miRNA Analogs.

Fluorescence was measured on Day 15 (540 excitation/590 emission) in cells transfected on Day 3 with the Dy547-labeled non-targeting miRIDIAN Mimic and Hairpin Inhibitor (100 nM). As shown in FIG. 9, there was a significantly greater fluorescence of cells transfected with the fluorescent miRNA analogs, even 12 days after transfection:

b. Reduction of Control Gene Expression.

To confirm successful transfection of pre-adipocytes, the reduction of expression of the control gene GAPDH (“housekeeping gene”) was measured 4 days (Day 7) (FIG. 10A) and 12 days (Day 15) (FIG. 10B) after transfection of pre-adipocytes with a GAPDH-specific siRNA. Cell lysates were obtained and RT-PCR was conducted using pure RNA obtained by Cells-to-Ct reagents. 91% and 86% knockdowns of the GAPDH mRNA expression were observed at Day 4 and Day 12 post transfection, both highly significant, as shown in FIG. 10.

c. Phenotypic Changes During Human Preadipocytes Differentiation into Adipocytes.

At the end of treatment (15 days in culture) cells were stained with Oil Red 0 for assessment of lipid content. As shown in FIG. 11, in the presence of medium without rosiglitazone, the preadipocytes show little differentiation into lipid-loaded mature adipocytes. In the presence of differentiation medium including 100 nM Rosiglitazone for 2 days followed by maintenance medium for 12 days (negative control), some differentiation into lipid-loaded mature adipocytes is noted. In the presence of 100 nM rosiglitazone throughout the experiment (positive control), most of the cells became lipid-loaded mature adipocytes. As an example, in the presence of 25 nM hsa-miR-30b mimic, about half of the cells became lipid-loaded mature adipocytes. The non targeting miRNA mimic and inhibitor showed patterns similar to the negative control.

d. Genotypic Changes During Human Pre-Adipocyte Differentiation into Adipocytes.

Profiling of mRNA changes occurring during the differentiation of human pre-adipocyte into mature adipocyte induced by rosiglitazone or miRNA analogs was performed by RNA-Seq technology. Small RNA sequencing (RNA-Seq) is a high-throughput next-generation sequencing platform which now allows transcriptome-wide profiling of all small RNAs, known and unknown, with no need for prior sequence or secondary structure information.

RNA samples were extracted from pre-adipocytes (pre-adipocyte negative control) and from pre-adipocytes cultured in the presence of 100 nM rosiglitazone (differentiation positive control) or 25 nM miRNA mimics or inhibitors for 12 days. RNA sequencing was performed on the Illumina Hi-Seq 2000 equipment. The results were mapped against Human Genome 19 (http://genome.ucsc.edu/). It appears that in the presence of a miRNA analog, between 313 and 449 mRNA are significantly differentially expressed in reference to pre-adipocytes. In reference to Rosiglitazone, the number of significantly differentially expressed genes is reduced between 111 and 216, thus suggesting common pathways of activation of adipocyte differentiation between miRNAs and the PPARG analog.

Regarding our 83 thermogenic activators and inhibitors, the expression of 73 of them is altered in the presence of rosiglitazone or miRNA analogs. The changes of mRNA expression of the thermogenesis targets in the presence of rosiglitazone (FIG. 12A) or miRNA analogs hsa-let-7a inhibitor, hsa-miR-1 mimic, hsa-miR-19b mimic, hsa-miR-30b mimic or control adipocytes are shown on FIGS. 12B-F, respectively).

Changes in mRNA expression of UCP1, 2 and 3 were also measured in the presence of rosiglitazone or miRNA analogs, as shown below in Table 14.

TABLE 14 Changes in thermogenic mRNA expression. mRNA Expression changes (log ratios) Agent UCP1 UCP2 UCP3 Rosiglitazone 15.70 263 0.26 hsa-let-7a inhibitor 2.23 173 0.65 hsa-miR-1 mimic 0.41 110 0.40 hsa-miR-19b mimic 0.18 33 0.26 hsa-miR-30b mimic 0.76 119 0.28 Baseline level in 0.02 1.35 0.30 pre-adipocytes

The expression levels of the three Uncoupling Proteins were low in pre-adipocytes. The expression of UCP1 was significantly increased in the presence of rosiglitazone 100 nM which was renewed with the culture medium every other day. The magnitude of UCP1 mRNA rise with the miRNA analogs was lower than with rosiglitazone, but one has to keep in mind the miRNA analogs concentration used (25 nM) and the fact that only one transfection was performed 12 days before RNA extraction. A major finding is the dramatic increase of UCP2 expression in the presence of rosiglitazone as well as the miRNA analogs. The expression of UCP3 did not change in any condition, as expected for a gene that is mainly expressed in myocytes. This increase in UCP1 and UCP2 expression suggests that administration of these miRNA produces adipocytes with greater potential for thermogenesis and thus are likely effective pharmaceuticals for the treatment of obesity and other metabolic diseases and disorders.

Furthermore, we looked at genes differentially expressed during pre-adipocyte culture in the presence of miRNA analogs. As an example shown on FIG. 16, an M-A plot was created to visualize the differences of mRNA expression between pre-adipocytes grown in maintenance medium and pre-adipocytes grown in the presence of hsa-miR-19b mimic. The x-axis is the mean gene expression and the y-axis is the difference between pairs in logarithmic scale. The red dots are the differentially expressed genes (up regulated above zero and down regulated below zero). The gray dots are the genes not differentially expressed between control and hsa-miR-19b mimic (up regulated above zero and down regulated below zero).

As an example shown on FIG. 17, in reference to pre-adipocytes cultured in maintenance medium only, the numbers of significantly differentially expressed genes in the presence of the miRNA analogs hsa-let-7a inhibitor, hsa-miR-1 mimic, hsa-miR-19b mimic and hsa-miR-30b mimic were respectively 406, 382, 370 and 433. A set of 127 genes was commonly upregulated by these 4 miRNA analogs (Venn Diagram, FIG. 17).

They include not only some of our 83 thermogenic targets like ALDH1A1, AZGP1, CEBPA, PPARGC1A, UCP1 and UCP2 highlighted in green, but also numerous genes involved in lipid metabolism and adipocyte differentiation, highlighted in yellow (Table 15).

TABLE 15 Set of 127 genes commonly upregulated by 4 miRNA analogs. ABCC6 ABCD2 ACACB ACHE ACSF2 ACSM5 ACSS2 ADH1B AIF1L AKR1C3

AOC3 AOC4 APCDD1 APOC1 AQP3 AQP7 AQP9

BBOX1 BHLHE22 C11orf87 C14orf180 C1orf115 C1orf95 C3 CA2 CADM3 CDO1

CFD CFHR1 CHI3L2 CILP CKB CKMT1B CLCA2 CLMN COL14A1 COL21A1 CPB1 CYB5A CYP4F12 CYP4F22 DARC DGAT2 DHCR24 DPT DTX4 EPHB6 FABP4 FADS2 FAM65C FMO1 FMO2 G0S2 GPD1 GPR109A GPR109B HAVCR2 HRASLS5 IGSF10 ITIH1 ITIH5 KCNE3 KCNK3 KIT KLB LBP LEP LGALS12 LIPE LPL LRRC4C LRRN4CL MAN1C1 MAOA MAOB MARCO MCAM METTL7A MGP MLXIPL MOBKL2B MOSC1 MVD NAT8L NKD2 PCSK9 PFKFB1 PKD1L2 PLA2G2A PLIN1 PLIN4 PLXDC1

PPL PPP1R1A PRKAR2B PTGDS QPRT RASL12 RNF157 S100B SDPR SELENBP1 SEMA3G SEPP1 SLC2A4 SLC2A5 SLC40A1 SLCO4C1 SMOC2 SNCG SPARCL1 SPRY1 SVEP1 TF TM7SF2 TMEM132C TMEM176B TMEM37 TNMD TPRG1 TRIL

A set of 60 genes was commonly downregulated by these 4 miRNA analogs (Venn Diagram, FIG. 18).

They include numerous chemokines genes and genes involved in cell proliferation and (Table 16).

TABLE 16 Set of 60 genes commonly downregulated by 4 miRNA analogs. ACTC1 ANLN ARSI ATOH8 AURKB BLM BRCA2 BUB1 BUB1B CASC5 CCL26 CDC6 CDCA5 CDCA8 CDH15 CENPF CKAP2L CXCL1 CXCL2 CXCL3 CXCL5 CXCL6 E2F7 ESCO2 FAM83D GABBR2 GREM2 GTSE1 HAS1 HJURP ID1 ID3 IER3 IL13RA2 IL6 IL8 INHBA IQGAP3 KIAA1244 KIF11 KIF14 KIF18B KIF2C KIFC1 KRT34 KRTAP2-1 MALL MMP3 NCAPH PHLDA1 PLK1 PPAPDC1A PTGS2 RELN SHCBP1 SLC17A9 SLC6A17 THBD TMSL3 TOP2A B. Differentiation of Human White Adipocytes into Brown Adipocytes.

1. Differentiation Protocol.

In order to assess the effect of miRNA analogs on human white adipocytes differentiation into brown adipocytes, human subcutaneous pre-adipocytes (SuperLot 0048 from 8 female donors, ZenBio, NC) were plated on Day 0 into 96-well plates and allowed to attach overnight in preadipocyte medium (DMEM/Ham's F-12 (1:1, v/v), HEPES buffer, Fetal bovine serum and Antibiotics). The next day (Day 1), the medium was removed and replaced with differentiation medium-2 (DMEM/Ham's F-12 (1:1, v/v), HEPES buffer, Fetal bovine serum, Biotin, Pantothenate, Human insulin, Dexamethasone, Isobutyl-methylxanthine, Proprietary PPARG agonist and Antibiotics. The cells were allowed to incubate for 7 days at 37° C., 5% CO₂. After 7 days (Day 7), a partial medium exchange was performed with AM-1 adipocyte maintenance medium (DMEM/Ham's F-12 (1:1, v/v), HEPES buffer, Fetal bovine serum, Biotin, Pantothenate, Human insulin, Dexamethasone and Antibiotics). The cells were allowed to incubate for an additional 7 days at 37° C., 5% CO₂. On Day 17, the cells were transfected with miRNA analogs (Dharmacon specific miRIDIAN Mimics and Hairpin Inhibitors) using the transfecting agent Dharmafect 3. All treatments were in triplicate. Post transfection, the negative control was maintenance medium only and the positive control was maintenance medium with 100 nM of the PPARG agonist rosiglitazone. After 2 days, medium was removed and replaced with fresh maintenance medium. The maintenance medium then changed every two to three days until the end of the treatment period (Day 30). At the end of the treatment (total of 30 days in culture) cells were processed for Phenotyping and Genotyping Screening.

2. Transfection of Adipocytes.

Transfection reagents are used to facilitate the penetration of miRNA analogs into target cells.

As an example, the extent of transfection efficiency we achieved in adipocytes with the transfecting agent Dharmafect 3 (Dharmacon, CO) is depicted herein. Transfection efficiency was assessed in two ways:

a. Measurement of Cellular Epifluorescence after Transfection with Fluorescent miRNA Analogs.

Fluorescence was measured on Day 30 (540 excitation/590 emission) in cells transfected on Day 17 with the Dy547-labeled non-targeting miRIDIAN Mimic and Hairpin Inhibitor (100 nM). As shown in FIG. 13, there was a significantly greater fluorescence of cells transfected with the fluorescent miRNA analogs, even 12 days after transfection:

b. Reduction of Control Gene Expression.

To confirm successful transfection of adipocytes, the reduction of expression of the control gene GAPDH (“housekeeping gene”) was measured 4 days (Day 22) and 12 days (Day 30) after Dharmafect 3 (Dharmacon, CO) mediated transfection of adipocytes with a GAPDH-specific siRNA. Cell lysates were obtained and RT-PCR was conducted using pure RNA obtained by Cells-to-Ct reagents. Efficient transfection of mature adipocytes (a cell type known to be difficult to transfect) was achieved with the transfecting agent Dharmafect 3. 54% and 73% knockdowns of the GAPDH mRNA expression were observed at Day 4 and Day 12 post transfection, both highly significant, as shown in FIG. 14.

c. Optimization of Human Mature Adipocyte Transfection.

As efficient transfection of mature adipocytes is known to be difficult to achieve, we tested eleven different transfecting agents and assessed the degree of reduction of mRNA expression of the control gene GAPDH. Human subcutaneous pre-adipocytes were plated in 6-well plates and differentiated for two weeks following the protocol described above. Subsequently, a miRNA mimic (50 nM) targeting GAPDH was introduced into the differentiated adipocytes using transfecting agents following their manufacters' protocol. The transfected cells were incubated for 72 hours with reagents and miRNA mimic, then switched to maintenance medium. Fourteen days post-transfection, RNA was isolated using RNeasy Mini kit and RT-PCR reactions for the control gene GAPDH and the reference gene 18S were performed in triplicate using 100 ng of cDNA per well.

The amounts of RNA extracted per well were very similar, except for the transfecting agents TransIT TKO and TransIT siQuest which may produce potential cellular toxicity in the conditions of the experiment (FIG. 19).

The cells transfected with Dharmacon 1 and siPORT NeoFX had significantly reduced levels of 18S expression and were excluded from the RT-PCR experiment analysis. Among the remaining 7 transfecting agents analyzed, the often-used transfecting agent Lipofectamine RNAiMAX led to a 66% reduction of GAPDH expression at day 14 post-transfection, Dharmafect 3 and Dharmafect 4 respectively produced 60% and 75% reduction of GAPDH expression (FIG. 20).

3. Phenotypic Changes During Maintenance of Human Adipocytes in Culture for Two More Weeks.

At the end of treatment (total of 30 days in culture) cells were stained with Oil Red 0 for assessment of lipid content. In the presence of medium without rosiglitazone from Day 16 to Day 30, the adipocytes appear loaded with large lipid droplets. In the presence of differentiation medium including 100 nM Rosiglitazone for 2 days followed by maintenance medium for 12 days (negative control), little change in appearance of the lipid-loaded mature adipocytes is noted. In the presence of 100 nM rosiglitazone throughout the experiment (positive control), the intensity of the red staining seems reduced. As an example, in the presence of 25 nM hsa-miR-30b mimic, the intensity of the red staining seems also reduced and the lipid droplets appear smaller.

The amount of lipids present in the mature adipocytes at Day 30 was measured with the fluorescent Nile Red Dye. As shown in FIG. 15, the highest fluorescence was noted in the adipocytes which were not exposed to rosiglitazone from Day 15 to day 30. A similar fluorescence level was noted in the cells which were transfected with the non targeting miRNA mimic and inhibitor. When the cells were exposed to rosiglitazone for two days, the fluorescence dropped significantly and was further reduced in the presence of rosiglitazone from Day 15 to Day 30. It appears that in the presence of the miRNA inhibitors tested, the level of fluorescence is within the range observed with rosiglitazone 2 day to throughout. In the presence of miRNA mimics, the level of fluorescence appears lower, an indication of lower lipid content.

Example 4 High-Throughput miRNA Target Screening by Luciferase Activity and qRT-PCR

High-throughput screening using luciferase reporter assay constructs are used to identify novel miRNA targets involved in thermogenesis.

Luciferase is commonly used as a reporter to assess the transcriptional activity in cells that are transfected with a genetic construct containing the luciferase gene under the control of a promoter of interest. SwitchGear Genomics has created a genome-wide library of over 18,000 human promoters and 12,000 human 3′ UTR regions cloned into an optimized luciferase reporter vector system containing SwitchGear's RenSP reporter cassette (GoClone™) as a component of the LightSwitch™ Luciferase Assay System. This modified form of luciferase greatly facilitates detailed kinetic studies, especially those focusing on repression, which might otherwise be obscured by reporter protein accumulation.

The multiple microRNAs-one mRNA paradigm was tested with the SwitchGear Genomic GoClone system, using UCP1 as the single thermogenic target gene. In order to explore the possible interactions between various huma miRNAs and the 3′UTR region, the 5′UTR region and the promoter/enhancer region of the human UCP1 gene in Hela and HepG2 cells, three reporter constructs were made:

-   -   1. A human UCP1 3′UTR construct containing a reporter gene         driven by a strong constitutive promoter (RPL10_prom) with a         2,218 bp 3′UTR fragment of the human UCP1 sequence cloned in the         3′UTR region of the reporter gene. The effects of a specific         miRNA mimic, inhibitor, or non-targeting control on this         reporter's activity are compared to those of an empty_3′UTR and         an Actin Beta_3′UTR to identify effects that are specific to the         putative UCP1 3′UTR construct.     -   2. A human UCP1 Promoter construct containing a reporter gene         driven by a 4,147 bp 5′UTR fragment of the human UCP1 sequence         that spans the Transcription Start Site and upstream region         covering the methylation region and the enhancer region of the         human UCP1 gene sequence. The effects of a specific miRNA mimic,         inhibitor, or non-targeting control on this reporter's activity         are compared to those of an Actin Beta Promoter to identify         effects that are specific to the putative UCP1 5′UTR construct.     -   3. A human UCP1 Enhancer Region construct containing a reporter         gene driven by a short minimal promoter from the HSV-TK locus         with a 601 bp 5′UTR fragment of the human UCP1 sequence that         spans the Enhancer Region of the human UCP1 gene sequence. The         effects of a specific miRNA mimic, inhibitor, or non-targeting         control on this reporter's activity are compared to those of an         empty 5′ Enhancer Region to identify effects that are specific         to the putative UCP1 5′ Enhancer construct.

In addition, miRNAxxx_3′UTR constructs were made. They contain the reporter gene driven by a strong promoter (RPL10_prom) with a perfect match to the target sequence of miRNAxxx cloned into the 3′UTR region of the reporter gene. The effect of a miRNA mimic, inhibitor, or non-targeting control on this reporter's activity can be compared to EMPTY_3′UTR and Actin B_3′UTR to determine whether a miRNA mimic's or inhibitor's activity can be reasonably detected in the experimental cell type. If the cell type has no endogenous expression of the miRNA in question, the addition of a mimic should knock down the activity of this reporter, and the addition of an inhibitor should have no significant effect. If the cell type has high endogenous expression of the miRNA in question, the addition of an inhibitor should increase the activity of this reporter, and the addition of a mimic should have no significant effect. The range of endogenous miRNA expression in Hela and HepG2 cell types is broad, so the synthetic target activity changes are likely to reflect this variability.

For each miRNA candidate (38 in total), the following conditions were tested:

-   -   miRNA mimic (specific)*8 reporter constructs in Hela cells     -   miRNA mimic (specific)*8 reporter constructs in HepG2 cells     -   miRNA mimic non-targeting control*8 reporter constructs in Hela         cells     -   miRNA mimic non-targeting control*8 reporter constructs in HepG2         cells     -   miRNA inhibitor (specific)*8 reporter constructs in Hela cells     -   miRNA inhibitor (specific)*8 reporter constructs in HepG2 cells     -   miRNA inhibitor non-targeting control*8 reporter constructs in         Hela cells     -   miRNA inhibitor non-targeting control*8 reporter constructs in         HepG2 cells

To the extensive list of miRNAs that may bind to the UCP1 sequence, 8 filters were applied (in addition to required binding to UCP1 3′UTR region) to reduce the number of miRNA candidates to be tested. These filters were length of binding sites, number of binding sites, binding to the 5′UTR region, chromosomal clustering with other miRNAs, intronic location, binding to the Enhancer Region, binding to the Methylation Region and proof of experimental evidence of a relation to UCP1. 38 miRNAs that met at least 3 of these criteria were tested (Table 17).

TABLE 17 miRNA with putative binding sites in the UCP1 gene sequence. # of Binding # of Exp. miRNA criteria length sites 3′UTR 5′UTR Clustering Intronic Enhancer Methylation Evidence 1 hsa-miR-130b-5p 7 11 3 + + 22 + + + 2 hsa-miR-328 6 10 4 + + + + + 3 hsa-miR-655 6 10 5 + + 14 + + 4 hsa-miR-19b-2-5p 5 10 4 + + X + 5 hsa-miR-26a-2-3p 5 10 7 + + + + 6 hsa-miR-367-3p 5 10 to 18 3 + +  4 + 7 hsa-miR-371a-5p 5 10 to 12 9 + + 19 + 8 hsa-miR-377-3p 5 10 to 14 5 + + 14 + 9 hsa-miR-378a-3p 5  7 to 13 19 + + + + 10 hsa-miR-382-3p/5p 5 15 2 + + 14 11 hsa-miR-421 5 10 5 + + X + 12 hsa-miR-515-3p 5  9 3 + + 19 + + 13 hsa-miR-620 5 10 7 + + + + 14 hsa-miR-941/2 5  9 5 + + 20 + 15 hsa-miR-1179 4 11 3 + + 15 16 hsa-miR-1302 4 10 5 + + + 17 hsa-miR-146a 4  9 to 10 8 + + + 18 hsa-miR-181c 4  9 5 + + 19 + 19 hsa-miR-203 4  9 1 + 14 + + 20 hsa-miR-331-5p 4  8 to 15 6 + + 12 21 hsa-miR-422a 4  7 to 14 6 + + + 22 hsa-miR-452 4  8 7 + + X + 23 hsa-miR-491-5p 4 10 3 + + 24 hsa-miR-501-3p 4 10 2 + + X + 25 hsa-miR-543 4 10 to 14 4 + + 14 26 hsa-miR-545 4 11 2 + + X + 27 hsa-miR-549 4 13 to 14 3 + + + 28 hsa-miR-643 4 10 to 14 9 + + + 29 hsa-miR-651 4 10 6 + + + 30 hsa-miR-654-3p 4  8 to 10 11 + + 14 31 hsa-miR-21-5p 3 10 to 14 2 + + + 32 hsa-miR-211-5p 3 11 1 + + + 33 hsa-miR-22-3p 3  9 5 + + + 34 hsa-miR-30b-5p 3 10 1 +  8 + 35 hsa-miR-325 3 7 to 8 11 + + + 36 hsa-miR-362-5p 3 10 1 + X + 37 hsa-miR-504 3  9 2 + + + + 38 hsa-miR-552 3  9 3 + + +

In these Luciferase reporter gene assay experiments, a miRNA candidate was considered to interact with UCP1 if both the specific miRNA inhibitor increases the luciferase signal and the specific miRNA mimic decreases the luciferase signal with an Inhibitor/Mimic Ratio≧1.5 and or/a p value<0.05. These selection criteria identify 9 miRNAs (miR-19b-2-5p, miR-21-5p, miR-130b-5p, miR-211, miR-325, miR-382-3p/5p, miR-543, miR-515-3p, and miR-545) (Table 18). A few more barely missed these selection criteria; they are miR-331-5p, miR-552, miR-620, and miR-1179.

TABLE 18 miRNA identified as regulators of UCP1 by luciferase reporter assay. # of Binding # of Exp. Cell Line miRNA criteria length sites 3′UTR 5′UTR Clustering Intronic Enhancer Methylation Evidence 1 Hela hsa-miR-130b-5p 7 11 3 + + 22 + + + 2 hsa-miR-328 6 10 4 + + + + + 3 hsa-miR-655 6 10 5 + + 14 + + 4 Hela + HepG2 hsa-miR-19b-2-5p 5 10 4 + + X + 5 hsa-miR-26a-2-3p 5 10 7 + + + + 6 hsa-miR-367-3p 5 10 to 18 3 + +  4 + 7 hsa-miR-371a-5p 5 10 to 12 9 + + 19 + 8 hsa-miR-377-3p 5 10 to 14 5 + + 14 + 9 hsa-miR-378a-3p 5  7 to 13 19 + + + + 10 HepG2 hsa-miR-382-3p/5p 5 15 2 + + 14 11 hsa-miR-421 5 10 5 + + X + 12 Hela hsa-miR-515-3p 5  9 3 + + 19 + + 13 hsa-miR-620 5 10 7 + + + + 14 hsa-miR-941/2 5  9 5 + + 20 + 15 hsa-miR-1179 4 11 3 + + 15 16 hsa-miR-1302 4 10 5 + + + 17 hsa-miR-146a 4  9 to 10 8 + + + 18 hsa-miR-181c 4  9 5 + + 19 + 19 hsa-miR-203 4  9 1 + 14 + + 20 hsa-miR-331-5p 4  8 to 15 6 + + 12 21 hsa-miR-422a 4  7 to 14 6 + + + 22 hsa-miR-452 4  8 7 + + X + 23 hsa-miR-491-5p 4 10 3 + + 24 hsa-miR-501-3p 4 10 2 + + X + 25 Hela hsa-miR-543 4 10 to 14 4 + + 14 26 hsa-miR-545 4 11 2 + + X + 27 hsa-miR-549 4 13 to 14 3 + + + 28 hsa-miR-643 4 10 to 14 9 + + + 29 hsa-miR-651 4 10 6 + + + 30 hsa-miR-654-3p 4  8 to 10 11 + + 14 31 Hela + HepG2 hsa-miR-21-5p 3 10 to 14 2 + + + 32 Hela hsa-miR-211-5p 3 11 1 + + + 33 hsa-miR-22-3p 3  9 5 + + + 34 hsa-miR-30b-5p 3 10 1 +  8 + 35 Hela + HepG2 hsa-miR-325 3 7 to 8 11 + + + 36 hsa-miR-362-5p 3 10 1 + X + 37 hsa-miR-504 3  9 2 + + + + 38 hsa-miR-552 3  9 3 + + +

Out of these 9 selected miRNAs, 3 appear to bind to the 3 regions of UCP1 which were studied (miR-21-5p, miR-211, and miR-515-3p); 3 appear to bind to 2 regions of UCP1 (miR-19b-2-5p, miR-130b-5p, and miR-325), and 3 bind to a single region of UCP1 (miR-331-5p, miR-543, and miR-545). All but miR-331-5p appear to bind to the 3′UTR region of UCP1 (Table 19).

TABLE 19 miRNA identified as regulators of UCP1 by luciferase reporter assay. miRNA UCP1 3′ UTR UCP1 Enhancer UCP1 Promoter 1 mir-21-5p X X X 2 miR-211 X X X 3 mir-515-3p X X X 4 mir-19b-2-5p X X 5 mir-130b-5p X X 6 mir-325 X X 7 miR-331-5P X 8 mir-543 X 9 mir-545 X

Further screening is performed by transfection of the promoter/3′UTR library into human adipocytes or adipose-derived mesenchymal stem cells in cell culture, followed by addition of miRNA agents (e.g., agomirs or antagomirs) to the cell culture. Measurement of luciferase activity and identification of mRNAs is performed 24 hours after transfection and addition of miRNA agents.

In order to confirm the results of the transfection experiments set forth above over a longer time frame, lentiviral transduction experiments are performed using lentiviral vectors containing the miRNA agents of interest (from System Biosciences (SBI) collection of miRNA precursors expressed in the pMIRNA1 SBI vectors allowing the expression of the copGFP fluorescent marker). Specifically, cells containing the promoter/3′UTR library are transduced with lentiviral particles at an MOI of 1:10 and GFP-positive cells are sorted by FACS, according to the supplier's instructions. The level of expression of the mature miRNAs and their targeted mRNAs is assessed at several time points (0, 3, and 6 hr.; 1, 4, and 7 days) by Taqman Quantitative Real-time PCR in control cells (HEK293 cells), Human Adipose-Derived Mesenchymal Stem Cells, Human Subcutaneous Preadipocytes, and Human Proliferating Subcutaneous Adipocytes. Pooling of RNAs from 5 different time points after transduction is optionally employed to reduce the complexity of the qRT-PCR based screening approach while preserving the detection sensitivity.

Example 5 Proteomic Profiling

Proteomic Profiling is also used to identify novel miRNA targets involved in thermogenesis.

Shotgun proteomics is a method of identifying proteins in complex mixtures using high performance liquid chromatography (HPLC) combined with mass spectrometry (MS). Transfected and transduced cells with miRNA agents and promoter/3′UTR library (as described in Example 4) are harvested and lysed to produce crude soluble (cytosolic) and insoluble (nuclear) fractions. Peptides are from these fractions are then separated by HPLC and analyzed using nanoelectrospray-ionization tandem MS using the isotopic labeling technique SILAC to quantify protein abundance. Spectra are searched against the Ensembl release 54 human protein-coding sequence database using Sequest (Bioworks version 3.3.1, Thermo Scientific).

To avoid missing low abundance proteins, a targeted proteomics approach is also employed to accurately quantify a set of proteins that are known regulators of adipogenesis, adipocyte differentiation and BAT function. Some examples include UCP1, KDM3A, PRDM16, PPARA, PPARGC1A, CEBPB, CIDEA, BMP7, COX7A1, SIRT1, SIRT3, DIO2, FABP4, ADIPOQ. These proteins are analyzed via ELISA based or Luminex based immunoassays using commercially available antibodies.

Optionally, the protein fractions are analyzed using Multiple Reaction Monitoring-Mass Spectrometry on a proteomics platform, whereby only one protein (e.g. UCP1) of the thermogenic pathway is accurately quantified using LC-MS-MS.

Example 6 Reconciliation of the Phenotypic, Luciferase/qRT-PCR, and Proteomic Datasets

The results of the in vitro experiments set forth in Examples 3-5, herein, are reconciled. Specifically, to narrow further the initial set of microRNAs, mRNAs and target proteins and pathways to a relevant yet manageable number of targets, the experimental data is integrated with Network Searches and Analyses Packages (DAVID, Ingenuity Systems IPA and ARIADNE Pathway Studio.

Global analysis of the results of the in vitro experiments set forth in Examples 3-5, herein, is performed the Business Intelligence tool TIBCO Spotfire. This allows for a visualization of the relationships between the miRNA agents and target gene.

Example 7 Animal Models of Obesity

Several animal models of obesity have been developed and validated (Kanasaki K et al., J Biomed Biotechnol., 2011:197636 (2011); Speakman J et al., Obesity reviews: an official journal of the International Association for the Study of Obesity, 8 Suppl 1:55-61 (2007)). The most commonly used are the Leptin Signaling Defects Lep^(ob/ob) and Lepr^(db/db) Mouse Models as well as the High-Fat Diet model in C57BL/6J mice (Wang C Y et al., Methods in molecular biology, 821:421-433 (2012). This diet-induced obesity (DIO) model closely mimics the increased availability of the high-fat/high-density foods in modern society.

A DIO mouse model is used for in vivo validation of the effectiveness of the miRNA analogs described herein for the increase in thermogenesis and/or the treatment of obesity and other metabolic disorders (Yin H et al., Cell Metab., 17(2):210-224 (2013)).

DIO mice are administered one or more of an hsa-let-7a agomir, hsa-let-7a antagomir, hsa-miR-1 agomir, hsa-miR-1 antagomir, hsa-miR-19b agomir, hsa-miR-19b antagomir, hsa-miR-30b agomir, and hsa-miR-30b antagomir. Rosiglitazone is used as a positive control. Food intake, blood metabolic parameters, body composition (body weight, body fat, bone mineral and lean mass, body fat distribution, body temperature, O2 consumption and CO2 production, exercise induced thermogenesis, cold induced thermogenesis and resting thermogenesis are measured in the mice prior to treatment and after treatment. A reduction in body mass or body fat or an increase in body temperature or any kind of thermogenesis indicate the in vivo effectiveness of the administered composition.

Example 8 Nucleic Acid Sequences of Human UCP1 and UCP2 Genes and Transcripts

The nucleic acid sequence of the 1,462 base pair (bp) transcript ENST00000262999 of the human UCP1 gene is as follows (Exons in capital letters) [SEQ ID NO: FROM 351-363]

SEQ Start End ID No Exon/Intron Start End Phase Phase Length Sequence NO: 5′ upstream ...gtcggttcaaaaaacagaaatcgggtttgctgcccggcggacaggcgtga 351 sequence 1 EN5E00001 141,489,959 141,489,758 — 0   202 AGAGCAAGGG AAAGGAACTT CCTCCACCTT CGGGGCTGGA 352 081761 GCCCTTTTCC TCTGCATCTC CAGTCTCTGA GTGAAGATGG GGGGCCTGAC AGCCTCGGAC GTACACCCGA CCCTGGGGGT CCAGCTCTTC TCAGCTGGAA TAGCGGCGTG CTTGGCGGAC GTGATCACCT TCCCGCTGGA CACGGCCAAA GTCCGGCTCC AG Intron 1-2 141,489,757 141,489,132   626 gtagctaggc agaggggtaa gacaa...tgttc tgcacctttc ttatttccag 353 2 ENSE00001 141,489,131 141,488,933 0 1   199 GTCCAAGGTG AATGCCCGAC GTCCAGTGTT ATTAGGTATA 354 009006 AAGGTGTCCT GGGAACAATC ACCGCTGTGG TAAAAACAGA AGGGCGGATG AAACTCTACA GCGGGCTGCC TGCGGGGCTT CAGCGGCAAA TCAGCTCCGC CTCTCTCAGG ATCGGCCTCT ACGACACGGT CCAGGAGTTC CTCACCGCAG GGAAAGAAA Intron 2-3 141,488,932 141,484,673 4,260 gtaagccgtg agcgttcctg ggagg...aataa ttttttttct ctctggatag 355 3 ENSE00001 141,484,672 141,484,472 1 1   201 CAGCACCTAG TTTAGGAAGC AAGATTTTAG CTGGTCTAAC 356 081759 GACTGGAGGA GTGGCAGTAT TCATTGGGCA ACCCACAGAG GTCGTGAAAG TCAGACTTCA AGCACAGCCA TCTCCACGGA ATCAAACCTC GCTACACGGG GACTTATAAT GCGTACAGAA TAATAGCAAC AACCGAAGGC TTGACGGGTC TTTGGAAAG Intron 3-4 141,484,471 141,484,366   106 gtaactaact tcaaaatggg tttta...acatt ttctttttt ttttccccag 357 4 ENSE00001 141,484,365 141,484,264 1 1   102 GGACTACTCC CAATCTGATG AGAAGTGTCA TCATCAATTG 358 081762 TACAGAGCTA GTAACATATG ATCTAATGAA GGAGGCCTTT GTGAAAAACA ACATATTAGC AG Intron 4-5 141,484,263 141,483,528   736 gtaacttccc atttcatata acaaa...gacc tgtttcatcg atccatttta g 359 5 ENSE00001 141,483,527 141,483,347 1 2   181 ATGACGTCCC CTGCCACTTG GTGTCGGCTC TTATCGCTGG ATTTTGCGCA 360 081763 ACAGCTATGT CCTCCCCGGT GGATGTAGTA AAAACCAGAT TTATTAATTC TCCACCAGGA CAGTACAAAA GTGTGCCCAA CTGTGCAATG AAAGTGTTCA CTAACGAAGG ACCAACGGCT TTCTTCAAGG G Intron 5-6 141,483,346 141,481,165 2,182 gtaagatatg atcttgtgta tctgt...cgaac gatgacatgc acttttctag 361 6 ENSE00001 141,481,164 141,480,586 2 —   577 GTTGGTACCT TCCTTCTTGC GACTTGGATC CTGGAACGTC ATTATGTTTG 362 081760 TGTGCTTTGA ACAACTGAAA CGAGAACTGT CAAAGTCAAG GCAGACTATG GACTGTGCCA CATAATCAGC TTCAAGAAAA TGATGTAACA TACCAGTGGG AATCTTGCTG ACTGGATCAT AAAAACAAAC AAAACTTATT CACTTATTTT AACCTAAAAA GATAAAGGAA TTTTGGCAGA GAATTTTGGA CTTTTTTATA TAAAAAAGAG GAAAATTAAT GCCTATTTCA TATAACTTTT TTTTTTTCTC AGTGTCTTAA GAAGGGGAAA GCAAAACATT CAGCATATAC CCTGGCAAAT GTAATGCAGA TAAGCTACTG CATTTGACCA TTTCTGGAGT GCAATTGTGT GAATGAATGT GAAGAACTTT AACATGTTTT AATTACAATT CCAACTGGTG GAAAAGAAAC TGAGTGAAAT GCAGTTTATA TTTATAAATA CTTAAAAATG AAGTTATTAA AAATATTAGT TTTTATTAAC CACAGTTGTC AGTTAATATA TTCAATAAAA GTATTGCTAA TACCTTTT 3′ aaagtttgtcttttgagatctatacctgggtgtaagagtcaagttcacta... 363 downstream sequence

The nucleic acid sequence of the 9,371 base pair (bp) of the human UCP1 gene (ENSG00000109424) is as follows (Exons are highlighted) [SEQ ID NO: 364]:

>chromosome: GRCh37:4:141479988:141490559:−1

The nucleic acid sequence of the 15,901 base pair (bp) human UCP1 sequence (gi|237858805|ref|NG_012139.1| Homo sapiens uncoupling protein 1 (mitochondrial, proton carrier) (UCP1), RefSeqGene on chromosome 4) is as follows [SEQ ID NO: 365]:

CTGTACAGCT CTCCGACAAT CCCACATCTA GATGCCAAGC TGAGGTTGGC ATTCTCACTA    60 ATTTGCTGTT ATAAATATTA AGCTATCATA AGCGTTAGCC TACATATGAC TCTTTCATAT   120 GTTAGTTAAT TATTTTAGGG TAGAAATCCA AAAGTGGAGT TACCAGAAGT GGATATAGAC   180 ATTCTGGCTG GGTGTGATGG TTCATGCCTG TAATCCCAGC ACTTTGGGAG GCAGAGGCAG   240 GCGGATCACT TGAGGCCAGG AGTTTGAGAT CAGCCTGGGC CAACACAGCG AAACCCCATC   300 TCTACTAAAA ATTCCAAAAC TAGCCAGGCA TAGTGGCACA TGCCTGTACT CCCAGCTACT   360 TGGGAGGCTA AGACACAAGA ATCGCTTGAA CCCGGGAGGG AGGTGGAGGT TGCGGTGAGC   420 TGAGATTGTG CCACCGTACT CCAGCCTGGG TGACACAGCT AGACTCTGTT TCAAAAAAAA   480 AAAGAAAAAG AAAAGAAAAA AATAGACTTT CTCTTGGCTC AGTGTATACT GCCAAATTGT   540 TTTCCAAAAA AATTGTGTCA ATGTATAACA CCATCACTAA TATAGTATTG ATATTATGGT   600 TATTACATTT TAAAATTCAT AATTTGTAAT TATAACATTC ATAATTTATT ACTATTTATA   660 ATATTAATGT AAATGTATAT TATATATAAA TGTTATAGTA ATTATAACTT TGGTAGTGAC   720 AAAGTATTAA TTTATTAGGT GAAGTATATG CTTTTTTATT AGTGATAATA AATATATCCT   780 CTCTCCCATT ATAAAAGTTT GTATTTCTTC TTTTAGAAAT TGATTCTTCT GTCATTTGCA   840 CATTTATCTG TATAATTATA ACAGGGTATT TCCCAGTGGT GGCTAATGAG AGAATTATGG   900 GAAAGTATAG AACACTATTC AAATGCAAAG CACTGTATGA TTTTTATTTA ATAGGAAGAC   960 ATTTTGTGCA GCGATTTCTG ATTGACCACA GTTTGATCAA GTGCATTTGT TAATGTGTTC  1020 TACATTTTCA AAAAGGAAAG GAGAATTTGT TACATTCAGA ACTTGCTGCC ACTCCTTTGC  1080 TACGTCATAA AGGGTCAGTT GCCCTTGCTC ATACTGACCT ATTCTTTACC TCTCTGCTTC  1140 TTCTTTGTGC CAGAAGAGTA GAAATCTGAC CCTTTGGGGA TACCACCCTC TCCCCTACTG  1200 CTCTCTCCAA CCTGAGGCAA ACTTTCTCCT ACTTCCCAGA GCCTGTCAGA AGTGGTGAAG  1260 CCAGCCTGCT CCTTGGAATC CAGAACTACT TTCAGAATCT TGAACTTCTG TGACCTCTCA  1320 GGGTCCCCTT GTGTGAAGTT TTTGACGTCA GCTTCTCCTG TGACCCTTAG AAGTCACTCT  1380 TGTGTCTAGC ACATCCCAGG TGCTCAGTCA CCATTGAACT ACAGTCATAC TATCTCCTGG  1440 CAAAGGCTCT TAACTGTCCA TGTTAGCCTG ATATTAATAT CCTGGAAGCT TATACTGTCG  1500 TTCTTCCTTC CAGGTTTAAA TAAGGCAGCC CCTTTATCCT GTCACAGGTC CTCTCTCCCT  1560 ACCTATCCTT ACCTGTTTTG GATAACAACC TTTCTTATTT CTAATAGATT TATTTATTTC  1620 TCACATTTCC TTCCCTTATC ATAGTTTTCC TCTCACTTTC TCCTCTAGTT TGTCATACTC  1680 TGGCTTTAAA ACATGCAAAC ATGTGCCTTA TGGGGAAAAA AAGACAATTT TAATTTACCT  1740 TGCTTCTTCT TTACAAATGT ATTGTGGCTT CTTCTTATAG TCCAAATCTA AAACTCTTTA  1800 CCCACCCACT GCCTTGAACT CCTTCCTCGT TGTGAAAGTA GGATGGGGCA AAGAGAGAAT  1860 GCATGCCCCT CCCAACTGCT CAAACAAGTA AAGGTGCTGT TACAGTTATC TTTTGCTACC  1920 TTAATACAAT AATTATTTTA TTATATCTCA CAATTTTATG GATCAGGAAT TTAGACTGGG  1980 CTCAGCTAGG CGATTCTTCT GCTTTACTGA CATCATAGGA GATCACTTGG TGGTATTCAA  2040 CTGTCAGGTA GGCTTATCTG GAGGGTCCAA GATAGCTGTA CTCTGGTGCC TGGTGCCTTG  2100 GTAAAGAGGG ATGATGATGT GGGGCCTCTC CAGCATGAAC AGCCTCAGAG AAGTTTGCTT  2160 TCTTACATGC TGGCCCAGGG CTCCAAGAGC AAATGTTGCA GTGAGTAAAG CAGAAGATAC  2220 AAGGACTTTT ATAATCTGGT CTCAGAAGCC ACATGGCATC AGTTCTGTAT TATTCTATTG  2280 GTCAAAACAT TCATAAGCCT GCCAGATGCA AGGGGAAGGC ATATGTACCC TCATCTTTTG  2340 ATGGGAGGAA TGTGATGGAT TTGCAATTAT GTTTTAAAAC TACTACAGAC AGAACCACTG  2400 AGAAAGATTC ATGGGTAGCT TTGGGGTGAG GACTGGGAAT TAACCTGTTG ATAGCAGAGG  2460 TTCACTAGAG TCAACAAGGA ATAAGGTCTC CTCTTGTACA CTTTAGTCAT ACTATACCAA  2520 CATTCTTAAC CACTGCTTAG CCATCAGCCT CACAACATAA CAACTCCATC ATAGTTGTAC  2580 TCCCTAAGAT CACCAACAAT GTTAGAGTCA AATCCGGTAG GTTTTTCTTT GTTTTTGTCC  2640 TCCTGACATT TTTTCTAAAC TTGACACTGG TCAGACCCAA TCTTTCTTTA ATCATATTCT  2700 TAAATACCAG TTCTATCACT GGATATGTTA CTGTTTCTTG TTCTCACTCT ACCTTTGACA  2760 AAGCCATTCT TTCCAGACTA TAACTCTGGG TCTGGGTCCC CCTATGGTTT GGCCCTTGAA  2820 TTCTTTTCCT AGTCCTATTT GACTAGCCCC ATTTTCCCGT GAAAAGCATG CCCCTTTCAT  2880 TGCATCCATA TCATGACTAC CAAATACCTC CTCTATTTCT TCCTCTTTTA GCATGTTAAA  2940 TGCAGCTTCC TAAGCTCTCT ATCTGGATAT CAACAGTATT CTCTCCAAAT AATTCTAAGA  3000 CTTTAAAAAT TGGTTTAATC TTCTTACCCC TAAAATCACC CCCCTTACCA ACTGCCTCAT  3060 GACAATCATT GGTACTGTCA CTGAGCTTGC AACCCATGTT CTTAAACATA GAGTAATCTT  3120 TGACTCCACA TCTAATCATT CATAAAGCTG TATTGTCTAT CAAATTAAAT CTGACATTTA  3180 TGTGAGAGCA CTTCATAGTC TGTAAAGCAC TACACAGGTG ATAACATGAA GCTACACTCA  3240 TAATGGATTT GCAGGCTCTG CTTCTCATTT GGCTTCTACA GCCTCATCCC TCACCAACTT  3300 CTTGCCCTAC CTCTCTCTTT CTTCCCCATC ACCCAATTTC CCAGTCAGTC AGGCCAACAG  3360 AATGCATTCT ATATACGCGA CTTGCTTTCC CCAACATCTT TGCCTGTATG CATGCCACTT  3420 ATTTGCCTCA GTTGATCTTT ATTTCAACAA GTGTTTGCAG AGGAGAAACC TCGCTGGCTC  3480 CTTCTCCTTT CTATTTTTTT TCAGAGGCTA CCCGTCAGGT CAACATTGCC TTTTTCAGGG  3540 AAGCTCTGCA AGCCTGACCT CCCTTGGAAG TGCCTTAGGA CTGGCTTCTT GCACAGTACA  3600 CAACCTTTAC TTATAGAGGG TTTGGAGATT ATTCTTTATT CATGTCTTAT TTCTCCTGCT  3660 CCTGGAGGAG ATGACTCTGA CTTCCACTGA CTCTTTTGGG GGGCTTAAGT CAGGGTTGAG  3720 TACCAGAGGC CCTAAATAGC TGGACGTGGA TTCTGGTAAT ATCAAATCCA TCTTTGGCTT  3780 AACTGAGAGG TTCTGAAAGC TGGGACCTGA CCTTGTCCAT TTCCCTCTTT CTCCAGTTTC  3840 CTATTATTTC CCACTGTTTT TTTTAAAAGT TTTTTGTTTT CTTAAGTTTT CACAAGAATA  3900 AACATTGAAA ATAAAATTTG CACAAAGATC GAACTAGGAA AGGCCACACA ACCAACACAT  3960 ATTACATCAT TATAGGTAAG TTAGCAGGGA GATTTCAGAC CTGGGCTAGC TCTGGAACCA  4020 CATTTTACAC TGTTGAAAAT AAAAGCTGGA GTACAGATGA CTTTCCCAGG TTCACAGAGT  4080 TGGTAAGCTG GAGAGCTGCA CCTGGAGCCA AGCAACCTGC CCTGTCCTTT CCACTGCACC  4140 CTCTAAGAAA TCTAATTAGA AGGAACAGGT GGTATCTCAT TTTGTACGGT GCTTTAGCAA  4200 TGTACTATTT GCTTTCTAGT GTGTCTATTG TCTCGTTTGA CATCTTCTCT CAAAAAGTGA  4260 TGAAACGAAA CGCTCTTTTT GACAAGTTCA GAGTGCTCTT GGTTCCTGTG TGGGATTCTT  4320 CCAAGTCTGA ATTTGGTAGT GGGAAGAGAA GGAATCCGGA GGAAGGAGGA TGAGAAGTTT  4380 AAAGGAGAGG AAAGGGAAGC AGAGAAGGCC GCAAGGTGCC TGCAAGATGT CTGGGGAGTT  4440 GGAGGAATGG AAGAGTGCCC CGCTCTTCCT TCTGGGAGAG CTCCAGCTAG GCAGAACCTT  4500 TCACCAAGGC TCTGATATCG TGCTGGTTTC CGAAAGCCCC AGCCGAAGGT GTGCAGCCAA  4560 AGGGTGACAG AAGGTGAGGC ACGTGCGGGG GCGCGGGTGC TGACCGCCGC GGTGCGCCCT  4620 CCCTCCGACG TGCGGTGTGC GGGGCGCAGA CAACCAGCGG CCGGCCCAGG GCTTTCGGGG  4680 AGCGAAGCAG GGCTCCCGAG GCACCGAGCG AGAATGGGAA TGGGAGGGAC CCGGTGCTCC  4740 CGGACACGCC CCCGGCAGGT CCCACGCCCG GGTCTTCTGA GACCTCGCGC GGCCCAGCCC  4800 GGGAGCGGCC CAGCTATATA AGTCCCAGCG GAAGACCGGA ACGCAGAGGG TCCTGCTGGC  4860 GCGAGGGTGG GTAGGAGGGG ACGCGGGGAC TCGGCCCCCA ACACCGCGCT CCGTCTGCAG  4920 CCGCCGCCTC TGCACCGCCG CTGCCCGGCG GTCGGTTCAA AAAACAGAAA TCGGGTTTGC  4980 TGCCCGGCGG ACAGGCGTGA AGAGCAAGGG AAAGGAACTT CCTCCACCTT CGGGGCTGGA  5040 GCCCTTTTCC TCTGCATCTC CAGTCTCTGA GTGAAGATGG GGGGCCTGAC AGCCTCGGAC  5100 GTACACCCGA CCCTGGGGGT CCAGCTCTTC TCAGCTGGAA TAGCGGCGTG CTTGGCGGAC  5160 GTGATCACCT TCCCGCTGGA CACGGCCAAA GTCCGGCTCC AGGTAGCTAG GCAGAGGGGT  5220 AAGACAAGGG GTCTCAGGAC AGAGGGGACG CTGTTGCGTG CATTCCATTT ATTCTCTGCT  5280 TTGGTGTAAC CACTGTTTCT AGGTAGGGTA GGTGACCTTC CAAAGCAGTC TGGCCTTGTC  5340 CCAGGGCTGG TGCTTTAGGA TGGGAAACTG GAACTTTTTC TGGGATTAGC TGAAGAACCA  5400 CCAGGGCCAC AGAGAATGGG TTGACCATGA CTACTACCAA ATTCTCCCAA AATTTAGGGT  5460 GCACTTAGTA TTTTAAGAGC TGAGAATATT GGCCTCTCCT GAGTTTACTA GTCAGGTGCT  5520 TTTTCCTTTC TTTGATTCTT CGGGGGTTCT GTCCTATCCT ACTGCCCTAG GGGTTCTGGA  5580 GAGTTCCTGG GGAGGGGGAT ATTCAAAATG TGCATTGTAG CCAGCCTCCC TCCATCTGCG  5640 CGTGAGCGAA CACACACACA CACACACACA CACACACACA CACACACACA CACACACGGT  5700 AGAGGGAGGT GGATGGAAGA GGAATGTTGC TGAGAAAAGA AACGGAAAAT AGGAACACAG  5760 GGGGAAATCT TGGCTTAAGA GTGAACTCAA TTTCGCTCCC TTCTGTTCTG CACCTTTCTT  5820 ATTTCCAGGT CCAAGGTGAA TGCCCGACGT CCAGTGTTAT TAGGTATAAA GGTGTCCTGG  5880 GAACAATCAC CGCTGTGGTA AAAACAGAAG GGCGGATGAA ACTCTACAGC GGGCTGCCTG  5940 CGGGGCTTCA GCGGCAAATC AGCTCCGCCT CTCTCAGGAT CGGCCTCTAC GACACGGTCC  6000 AGGAGTTCCT CACCGCAGGG AAAGAAAGTA AGCCGTGAGC GTTCCTGGGA GGGGCAGAAA  6060 AGCCTTGGGC TCCGCTCTGT TCCAAAAAGT GTAACACACA GAGGAGTGGT TTTCATAACA  6120 AATTGGCGAG AAAACATTCA TATTTGAACT CTCCCTTCCC CAAACATTAG CTCATTGTTC  6180 ATAGAAAAAA GTATGCAAAA TCGATTTTTT AGATGCAGAT ATATACTTGT AAAGGTCACC  6240 CAGTCATGGA AGTTTTGTGC CCAGTTTGGA TCTCCATCTG GAGAATATGG GTGGGCTACA  6300 GAAAAATGTT TAACTTAAAG TTCTCCAAAG AGGGAAGTAT ATCAGAAACA TCTATGGAGC  6360 TTGTCAGAAA TCCAAACGAG GACTACCATG GTCCTCTGAG TCTGAATCCT CAGGCTAGAG  6420 ACCAGAGTGT CTTTCCACAA GCTTCCCTCA TCATTTGTGT ATGCAACAAA GTTCAAAGCC  6480 TTCTGTTTGA AGCAAAGAAA GCCAGACTTT GTGAAGAGAG TTGAAAGGAC AGGAAAAGAC  6540 ATATTTCCTC TTAAGAGGTT CCTCATCAGG TCCAGGAAAG ACCAGAGCAG AAAAAGTGGA  6600 CGAATGCTGC AGGGAGTTTG TTTAGGGGAA AAAGAAAAGG AAACATATTT CCTGAGTGCC  6660 AGTGCACTCT AAGAATTCCT GTCACTTTAG GTAGCATTTA TTTGAGGGCT TAACTATGAA  6720 CCAGACATTG TTCTAAGTGC TTCAGATACA TTATAACTGG AAGGGTATTA GTACCATTAT  6780 CCCTTGGCAG ATGGGAAAAC TGAACACAGA GCAGATTCAT CACTTGCCCA AGGTCACACA  6840 GCTGGGAGGG GGCAGAGCCA GGGTTCAAAC CCAGGCAGTC TGGCCTCGGA CTCCAGGCTC  6900 CTAACCCTGT TCTCTACTGC CTTCTGCACT TCTCATATGA TTCTGCCCAT CATTCAAACC  6960 GCACAACACT GCTGTGAGTA AAAAGTGTTA GCCGAATATC AGGGTAGTTA AGTAACATGC  7020 ACAAAATCAC ACAGCTAATC AACATCAGAG GCACTTTCAT GTGGAGTAGA CAAGCCAGAG  7080 AGAAGATGTG CTGATGGCAC AATGAATACA TTAAGTGAAA TCCACCTTGT AGATTTCATC  7140 ATTTCTGCTG TGAGTAACCT TCAATACTAT AATTTTATGG GATAATTTAT AAATGTTGTC  7200 TATACAAATA TATAAGTTAT ACTTATCCAC ACAAGTACTT TCAAAGTGAA GATAAAGTCT  7260 GGATGTTACT AGATCAAAAC TGCATTTTTT TATTTATAGA TGTAGCAAGA GAGGAAACAC  7320 AAAGGAGGTA AAGCTGCCCG TTCAGGTGGT TTTCTTCACA GATTGACTGT TCTACCAATT  7380 GTTGTGGACT TTGGGCACCA AATTAATAGG ATATATGTTG GCAGTGTTCT ATGTTATATA  7440 GATTCAGTTT ATTTAGTAGG CTTTATTGAA CTGCCATGTG CCAGTAACTA TGTTAGATGT  7500 TTAGATGGCA GATGTGTCTC TAGACAGAGC TTACAGTTGA GAGTATGGGT TGTGTGGGGA  7560 GAAGTGAATA GATGACTATA TTCCATGATA CATGCTGTAT TACAATACAG TCCTACTTCA  7620 CTTAACGATG GGGATACATT CTCAGAAATG AGTTAGGAGG CAAATTGGTT GTTGAATGAA  7680 CATCACAGAG AGCACTTACA CAAACCTAGA TGGCATAGCC ACACCTAGGC TATATGGTAT  7740 AATCTATTGC TCCTAGGCTA CAAACCTGTG CAGCATGTTG GTATTGAATA CTACAGGCAA  7800 TTGTTACATA AAGTTAAGTG TTTGTGTACC TAAAAATAGA AAAGGTAATG CATTACACTA  7860 CAGTCTTATG GGGCTGGGAT GTCACTAGGT GATAGGAATT TTTCAGCTCT GTTCTAATCT  7920 TACGGGACCA CCATCATGTA TGCAGCACAT GACTAACTGT AATTACAAGA TGGTGGCTAT  7980 ATTAAACAGA ACTACTTAAG CTAGCCATGG AGGTATGGTC CGTGAGATTT TCCTGAAGAA  8040 TTAACGTCTG GATCAATTCT GGAAGGGCCA GCAGGAGTAC TCCAGGCAAA GGGGTGAGAA  8100 AGGAGCTTCC AAGTAGAGTG AAGGTCATGT GCAAAGACTC AGTGAGGAGT CGAGTGAACA  8160 TAGCACAGGG AGGACATGTT GGTGAGGAAG GAGGGGTGAA GCCACAGAGA CAGGAGGGAG  8220 CCAGATGACA GAAGGCCTTG CAGGCGGTGC TAAGGAGTTT GGATTTTATC CTTACAGTGG  8280 TGGGAAGTCA TTGTAAAAAT ATTAAGCAAG GGAGTGGCAT AAACAATTTA CATTTTCAAA  8340 AGATCACTTT GGCAGCAGAT AGAGTATATA TGTAAAAGGA GTAAGAAAGA GGTAAGTTAG  8400 AAAGCAAGAA ATGATCAGGG TATGCCCTAA AACACTGGCA ATAGGGAAAA AGAGATGTCA  8460 ATCAGAAAGA TTGAGAAAGT ATAATTGAAT TGACTTGGTG AACAAATAGA AGTAAGGCAT  8520 AAGGGACAGG TAGAAATATG AGATGACTTC CAAGTTTCTG TTTAAAGATA CCCTTTATTG  8580 AGAGAGGATG TATAGAAGCT GTCTTAGGGG GAAGACAAGA AATTTGGTTT AGGCCATGTC  8640 AACAGGTAAT GGCCAGTAGG CACATGATTC AGTTTATTTA GTGGGCTCCT TTTAGGAGAA  8700 AATCTGAGCC AGATTCCAGG AAGTCACAGC AGGGACTACC AATAGGGTCA AACAGCAGAG  8760 AGTGTGGAAA GGACTGAAAA GTGATCATTG TACATAACAA ATAGAAGCTC ACTGATTTTC  8820 TAGCAAAAAC ATCTTCAGCA GAGTAGCGTG GTATAAGCTA TATTGTAGGG GACTGAGGAA  8880 GAAATGGGCT CTGAGAAGTA AAGACAAACA ATATGTTTTG TAAATAAATT TCTTTTAGTT  8940 CTTAAAAAAA AAGCCTCTTT TCCAGCTTGA TTGGGAAGTG AAGAGAGGGA TTTGAAAGTT  9000 GGAGATTGGA GGATAGGATG AGTACATCAA GATACACTAC GTTGTAGTGC AGTGCATTAC  9060 AAATGTGAGC TAAAAGTGAA GGCATTTGTA ATCATATGAT ATTGCTAATT AAAAGACAGC  9120 TGTCAGTCAT ATGCCCAGCT CCTGGTAAAG CATGATGAGA AGAGTACAAT CATGGTAGTG  9180 ATTTAAAAAT TGCTGCCAGT TTTGTGGATT TTCTTTATGC TAGACAGTGT AAGCTCTTTA  9240 TCAATATTAT TTAACTCACA CAACTCTAAG AGGTAGATAT TATTATCCCT TTTTGACAAA  9300 TTAGGAAACA GAATTATAAT GACTGAGAAA GTCTCTGCTG AGTAAATGTT ACTGAACCTT  9360 AATTTTATGT TTACTTAATG ATAGAAATGA ATATTGGGCT TCAAGACTAT TTGTACTTAA  9420 TGAAATCTGT CTTGAGCAAC ATAAGCTATT TTTTTCAAAA TTTTAAGACA AAAATCACTT  9480 TCTTCTCTCC TGTCTTCTTA TTTTTGTTCC CTTCACATGT TGTAGCCTAA CACTACTTGA  9540 TGGCCCATTT TGGTGCAGTT TGTCCACTGG GCTTCATCTA AGGCCACCAA GTCCCATAAT  9600 TAACATGATC ATTCGTGGGA GAAAGATCAA GCCTCATTGG TGATGGGTGC CTCCTCACAG  9660 TCGGATAATA CTGAAAAGAG AGCTAAATGT GGGAAAGAAC CAAGTTGAAC ACAGGAAAGA  9720 ATCAGGCCAC TGTGAAAATA AGCATTGTGT TTTCTTGTTC CTTGAAAGTC TTCATTTTTA  9780 AAAAATTTCA GACACCTGAA GTTTTCTAGC CTTACTCTGA GTTGACGCAC ATTTAGTACA  9840 TGATCAACAC ATAAACAAGC ATTAGAGAAA TAGAAAAGCT GTAAGAATAC AAAAATATGG  9900 GCCAGGTGGG TGGCTCATAC CTGTAATCCT AGCACTTTGG GAGGCCGAGG CAGACGGATC  9960 ACCTGAGGTC AGGAGTTCAA GACTAGCCTG GCCAATATAG TGAAACCCTG TCTCTACTAA 10020 AAATACAAAA CTTAGCAGGC TGTGGTGGCA CGTGCCTATA ATCCCAGCTA CTTGGGAGGC 10080 TGAGGCAGGA GAATCTCTTG AACCCGGGAG GCGGAGATTG CAGTGAGCCA AGATCACACC 10140 ACTGCACTCT AGCCTAGATA ACAGAGCAAG ACTCCATCTC AAAAAAAAAA AAAATACAAA 10200 AATATGAACC ACTGAAAATT AAAAAGACAT GCATGCATTC TAGGTCTTTA ATTTTTTTTC 10260 TTAATAATTT TTTTTCTCTC TGGATAGCAG CACCTAGTTT AGGAAGCAAG ATTTTAGCTG 10320 GTCTAACGAC TGGAGGAGTG GCAGTATTCA TTGGGCAACC CACAGAGGTC GTGAAAGTCA 10380 GACTTCAAGC ACAGAGCCAT CTCCACGGAA TCAAACCTCG CTACACGGGG ACTTATAATG 10440 CGTACAGAAT AATAGCAACA ACCGAAGGCT TGACGGGTCT TTGGAAAGGT AACTAACTTC 10500 AAAATGGGTT TTATAACCAC CAAAGCACAT ACATACAACT AGCAACTTAT TGTAAAGTAG 10560 AGTTAATAAA CATTTTCTTT TTTTTTTTCC CCAGGGACTA CTCCCAATCT GATGAGAAGT 10620 GTCATCATCA ATTGTACAGA GCTAGTAACA TATGATCTAA TGAAGGAGGC CTTTGTGAAA 10680 AACAACATAT TAGCAGGTAA CTTCCCATTT CATATAACAA ACAGGTCTGC ACCTTTAGAA 10740 GTTCATCTTG GAGCTTCTGC AGCCACCTTA TACTCAATCT CTTAACTCCA ATAGTTTTCT 10800 CTTTTTAAAA ATTAAGTAAT TTTGAACCAT ATATAACTTT GTGAGAAGCA GGAAAAGACC 10860 AAAATATTAA GTTTAAGAAG TTTTGCCACA ACAAAAATAT TTTGCAACAA AAATAACAGG 10920 CAATTTCATG TCAGCATTAT TCTCATTTAA TACTAATATA TGGGACTTTT GTTAGAATCT 10980 TATTCTTTAT ACAGCAGAAT TCAGGAGGTA AGTCCATCCT GCATACTATA TCCAAAAGAT 11040 CTAGTTATAA AAGGAGCTTA TCAGTGGTCT CATCCAAAAA GTAATACCAT AAGATAGGTT 11100 CTTAAAAATA ATATTCTAAC AACTTCTAGA GACATTGAAA TTTCCCTTAT TTCAATAAAA 11160 AAGTATTAGA TGCTCATATA TTAGGCATTA TTACAGGCCT TAAAGGCACA GAGGAAACTA 11220 ACAGTTTACT TTCCTAAAGT GTTAACAATC TATTAAGCCA TTTACTCTTT ACCTTCTTTT 11280 TCTAGTGCAA TACCTTTCTT ATTTTATTTT ATTTATTTAT AAGACATCTT CATTGACCTA 11340 CTGTTATCAA TAGGTTTATA AAGATATGAC AGATAACTAA ATTGCAAGCC CCCAAAAGTC 11400 TGATGTTGAC CTGTTTCATC GATCCATTTT AGATGACGTC CCCTGCCACT TGGTGTCGGC 11460 TCTTATCGCT GGATTTTGCG CAACAGCTAT GTCCTCCCCG GTGGATGTAG TAAAAACCAG 11520 ATTTATTAAT TCTCCACCAG GACAGTACAA AAGTGTGCCC AACTGTGCAA TGAAAGTGTT 11580 CACTAACGAA GGACCAACGG CTTTCTTCAA GGGGTAAGAT ATGATCTTGT GTATCTGTAA 11640 TGTGTTCTGG CTGTCTGTGT GCTTTGGGAC ACTCTCATGT CAAGCAACCG ACATTTAGCT 11700 TACAAGCCTT AGTATATTCA TATACTTAGT ATTGACTTTT CCTTGCCACA GATTTCTCCA 11760 ATCCACCAAT TCCACTGTGC CAGAAAGTAA AAAGCCATGA TATTCAAATT TTCTCAACTT 11820 TGATCAAAGG CTCATTCAAG ACCAGTGCCT TTTCCACTGG TCCCAATCTA CTGGAAATGC 11880 AGACAGTATT TTGCCTTCTC TGGGCAAGAA AGTTATAAAG TAGAGGGAAA TCATAATAGA 11940 GAGCTATGAG AGAACAAGAT TTGATTTGAT TTAATTTGAT GGACTCAAGT TTTAACATTG 12000 TAAAACTAGA GATAAGACAT CACCACCAAT CTAGAAAAGT GATGCAGAAA AGTATTTGAT 12060 TTGGGTAATT ATTACACTCA CCTAGAAACA AGTGTTGTGT AATAGATTAC ATATTTCCAT 12120 AATGCAATGT TGTATCAGAA ACTACCTTCC TAAGAAAATA TAGTATGGGC TCGGCGTGGT 12180 GGCTCGCACC TGTAATCCCA GCACTTTGGG AGATGGAGGC AGGAGGATCA CTTGAGCCCA 12240 GACTGGGCAA CAAAGCGAGA CCCTGTCTCA ACAAAAAATT TAAAAATTAG CTGAGTGTGG 12300 TGGCACGCAC TGATGGTCCC CTCTACTTGG GAAGCTGAGG CAAGAGGATC TCCTGAGCCC 12360 AGGAGTTCAA GGTTTCAGCG AGCTATGATT GTGCCACTGC ACTCCAGCCT GGGAGACAGA 12420 GCAAGTCCCT GTCTCAAAAA AGAAGAAGGA GAAGGAGGAG AAAATACAGT ATTAAGTAAT 12480 CTGTCAATAT ATTCCACAAG GATTACACTA GTGGTTTAAT AATAAAATTA TATTACCTTT 12540 TTAAATTGTA AGGCCATTCC TCAAGCTTTA TAAATTAAGC ATGAATGCAT CATACACATT 12600 TTATAAAAAG TTCCAACTCA TCATAATCTG TACTTATGAT ACATTAATAC AAATGAAGTT 12660 CATTATAAAA TTAACTTAAA ATGGATATAC CAGTTATTAA ACCATTAACC ATTTAATAAT 12720 TTTATTTTTT TCAAATTTAA AAACCTTTTG GGGAAGAAAT ACTACAACAT GGATGAACCT 12780 TGAAAACGTT ATGCTAAGTG AAATAAGCCA GACACAAAAG GACAAATACT GTATGATTAC 12840 ACTTAAATGA GGTACCTAGA GTAGTCAAAT TCATAGAGAC AGAAAGAATA GAAGTTACCA 12900 GGGGCTGGAG GTAGGAAAAA ATGGAGAGCT GTTTAATGGG TAGAGAGTTT CTTTTTGGGG 12960 TGACAAAAAG GTTCTAGAGA TGGATAGTGG TGATGGTTAC ACACAATGTG TGTGTACTTA 13020 ATGCTACTGA AATGTAATTT TATGATTTTT TTTTTTTGCA GCAAAATACC CCACATTGGG 13080 AAGTGAAGAG AAACATGTTA AGAGACTTGA AGGAAAAAAA TTGGGGCAGA GGGGTGTTTT 13140 TTATAGGTTA AACAATAAAA GCCATTTAAA CAGTAACAAT TTCTCTAAGG ACAAGAATCG 13200 TCAAGATTGA GACAGCACTG ATTTCTTGAC TCTACTCAAT ACTTCTTTGG TTTCTCTTCT 13260 TCCTTCCCCC TTCTAATAGT TTCCTACCTC CCATTCAGAA AGCAAAGCAA AACAAGCAAA 13320 AATTCCCCCT TCCCTCAAAA AAGGAAAGAG TTTTTGAAAA AGTTCATGTC AGTGAAGAAA 13380 AGACATGTTT TGGGAGTGAA GGATATTTGT GGATTTGTAT AGATGTGATC ATCAGGGCTG 13440 TGTTGTTTTG AAGTAATATA GGACATCTAG AGGAAAATTT ATTTTCAGCA GAGGAGGGAA 13500 AGATGAAGAG TAGGTACTTT TAAGCATCTT CACTTGAGGA GTGGCAAAAT GAGAAGCATA 13560 ACCTGCTATA ATCACTTTAA GAATTTCAGG CTGAGTGTGG TGGTGCAGTC TCTAGTCCCA 13620 GTTACTCCAG GAGGCTCAGG TGGGAGGATC ACTTAAGCCC AGGAGCTCGA GGTTGCAGTG 13680 AGCTATGATT ACACTACTGC ATTCCAGCCT GGGCGGCAGG GTGAAGCCTC ATCTCAAAAA 13740 TTAAAAAAAA AAAAAATCAA ACAAATTAAT CGAACGATGA CATGCACTTT TCTAGGTTGG 13800 TACCTTCCTT CTTGCGACTT GGATCCTGGA ACGTCATTAT GTTTGTGTGC TTTGAACAAC 13860 TGAAACGAGA ACTGTCAAAG TCAAGGCAGA CTATGGACTG TGCCACATAA TCAGCTTCAA 13920 GAAAATGATG TAACATACCA GTGGGAATCT TGCTGACTGG ATCATAAAAA CAAACAAAAC 13980 TTATTCACTT ATTTTAACCT AAAAAGATAA AGGAATTTTG GCAGAGAATT TTGGACTTTT 14040 TTATATAAAA AAGAGGAAAA TTAATGCCTA TTTCATATAA CTTTTTTTTT TTCTCAGTGT 14100 CTTAAGAAGG GGAAAGCAAA ACATTCAGCA TATACCCTGG CAAATGTAAT GCAGATAAGC 14160 TACTGCATTT GACCATTTCT GGAGTGCAAT TGTGTGAATG AATGTGAAGA ACTTTAACAT 14220 GTTTTAATTA CAATTCCAAC TGGTGGAAAA GAAACTGAGT GAAATGCAGT TTATATTTAT 14280 AAATACTTAA AAATGAAGTT ATTAAAAATA TTAGTTTTTA TTAACCACAG TTGTCAGTTA 14340 ATATATTCAA TAAAGTATTG CTAATACCTT TTAAAGTTTG TCTTTTGAGA TCTATACCTG 14400 GGTGTAAGAG TCAAGTTCAC TAGAATACAA GACTGCCCAA TAGCAAATGC AGGTCTTTAG 14460 AATCATAGGC ATGAACCTAC TCTGAATGTT ATTAGTATAG ATTTTTAATG TTTAGAGTCC 14520 AGATTTGATG ACATCTCTAA CAACTTCTAA TCTAAGACAC TATATTCATT TTGGCAGGAT 14580 TGCTACTAGA GTCTTGGTAT CTGTGCTAGC ATCACATAAT TTTAGAGCTG GAGGGTACTT 14640 CTGGGAAGAC AGAGGAACAG TTTGAGATTC CTACTGAGAT GAAAACGAAT CTTCATGGAA 14700 TCTTTCAGCA AAGCCAAATT CAAATTCATC ATTAGCACCT GTAGTAACCT TTTCAATGCC 14760 TACAAACTGC ATGCAGAAGA GATAGGGAAA CAGTAAAACA GATATTAAAA GAAGTTTTTA 14820 AGACAAAGCC CAGCCTGATT TTAAGCTAAA TCCAAGGATT GGCAGCTTGG ATGAGCAGGA 14880 AGGTTACAGG CTGCCAGACA TCATTCTAGT TCTGTTTTAA TCAACTCCAT GTTACATTTA 14940 CTATCAGGGA TTCTCACCTC ACCCTCATGC ATGTCTTCCC CATTCATTAC CCGCAAAAGT 15000 GTCTTGTAGC AGATGTCTTC TGTGTCCCAT ACATACCATT TTGCTCTTTA GTGCTTGCTG 15060 GCCTGACTTC CTATTGTCAT GTCAGCATCT GCCCTTTTTA GGGTCTCTGG CCACCAGAGC 15120 CAGCTTTACT CACCTGTGCA TGGCATTCTA GAAGAGCAGC AGGGAAAATA ACACAGCCCC 15180 AGTGCAGCCC TTAACCACCA ATAACTGGTA GTAGTTGGTG TACAAATATC TCAGTTCCCT 15240 CAACTGTCAG GTGGAATACC GCTGAGGGAT CAAACTCTAG TAACACACAG TAGTGTTTTG 15300 CTTACTATGG TTAACTAAAA AATCACAGGG TCTTCATGCA TTTGGAAAGG ATACTTTATT 15360 TCTTACAAAG GGTTACAGCC TACAAGGTGG TCATTCTGCA GGCTAGAAAG CGTAACCTCC 15420 AGCAAAGACC GGAGGCAGGC ACTTCTAGGG AAGGAAGAGT AAGACAGAAA TTTAAATTGA 15480 ATGGGTTGGC CAAGTATACA TATTCAACAG GCTACAGGTG GATTCATGAA TATTCATGAA 15540 GGCAGTCCTG ATGCATGCAT GTTACACCTT GGGGTGGAGG CTTAACATTT AAATGTATTA 15600 CAGTTAGGCC CTATACATGA AAAGGTGAAG CAGTAACACG AAGGCACACA ATGCACCATT 15660 TCTGTAAACA GGCCAGAGCC AGTTCACAGT GGTTGGTCTC TTATCATGAG AAAGCTACTA 15720 AAATCCTCTT GTCCAGTTAA AACTGTAGTT ATGGCTGGTG GAAAATGGGC TGGAGTCAGT 15780 CAACACTTGG TGAAGCTGCA GTTGCTTCAG ACACTCAAGG CCAGTGTTTG TTTAGCTGCT 15840 CGAGAAAAAG AAAAATCTTG TGGCAGTTAG AACATAGTTT ATTCTTTAAG TGTAGGAGTG 15900 TGTGACTTAA //

The nucleic acid sequence of the 2,113 base pair (bp) transcript ENST00000310473 of the human UCP2 gene is as follows (Eight Coding Exons in capital letters) (SEQ ID NO: 366-382):

SEQ Start End ID No Exon/Intron Start End Phase Phase Length Sequence NO: 5′ upstream ...aatcgacagc gaggccggtc gcgaggcccc agtcccgccc tgcaggagcc 366 sequence 1 EN5E00002 73,694,352 73,693,766 — —   587 AGCCGCGCGC TCGCTCGCA GAGGGTGGGT AGTTTGCCCAGCGTAGGGGG 367 287650 GCTGGGCCCA TAAAAGAGGA AGTGCACTTA AGACACGGCC CCGCTGGACG CTGTTAGAAA CCGTCCTGGC TGGGAAGGCA AGAGGTGTGT GACTGGACAA GACTTGTTTC TGGCGGTCAG TCTTGCCATC CTCACAGAGG TTGGCGGCCC GAGAGAGTGT GAGGCAGAGG CGGGGAGTGG CAAGGGAGTG ACCATCTCGG GGAACGAAGG AGTAAACGCG GTGATGGGAC GCACGGAAAC GGGAGTGGAG AAAGTCATGG AGAGAACCCT AGGCGGGGCG GTCCCCGCGG AAAGGCGGCT GCTCCAGGGT CTCCGCACCC AAGTAGGAGC TGGCAGGCCC GGCCCCGCCC CGCAGGCCCC ACCCCGGGCC CCGCCCCCGA GGCTTAAGCC GCGCCGCCGC CTGCGCGGAG CCCCACTGCG AAGCCCAGCT GCGCGCGCCT TGGGATTGAC TGTCCACGCT CGCCCGGCTC GTCCGACGCG CCCTCCGCCA GCCGACAGAC ACAGCCGCAC GCACTGCCGT GTTCTCCCTG CGGCTCG Intron 1-2 73,693,765 73,692,678 1,088 gtgagcctgg ccccagccct gcgcc...actct ctgcctttgc tcacccacag 368 2 ENSE00001 73,692,677 73,692,521 — —   157 GACACATAGT ATGACCATTA GGTGTTTCGT CTCCCACCCA TTTTCTATGG 369 184362 AAAACCAAGG GGATCGGGCC ATGATAGCCA CTGGCAGCTT TGAAGAACGG GACACCTTTA GAGAAGCTTG ATCTTGGAGG CCTCACCGTG AGACCTTACA AAGCCGG Intron 2-3 73,692,520 73,689,523 2,998 gtaagagtcc agtccaagga agagg...tgggg cttttctcct cttggcttag 370 3 ENSE00001 73,689,522 73,689,298 — 0   225 ATTCCGGCAG AGTTCCTCTA TCTCGTCTTG TTGCTGATTA AAGGTGCCCC 371 184370 TGTCTCCAGT TTTTCTCCAT CTCCTGGGAC GTAGCAGGAA ATCAGCATCA TGGTTGGGTT CAAGGCCACA GATGTGCCCC CTACTGCCAC TGTGAAGTTT CTTGGGGCTG GCACAGCTGC CTGCATCGCA GATCTCATCA CCTTTCCTCT GGATACTGCT AAAGTCCGGT TACAG Intron 3-4 73,689,298 73,689,142   156 gtgaggggat gaagcctggg agtct...tagct accctgtctt ggccttgcag 372 4 ENSE00001 73,689,141 73,688,931 0 1   211 ATCCAAGGAG AAAGTCAGGG GCCAGTGCGC GCTACAGCCA 373 252503 GCGCCCAGTA CCGCGGTGTG ATGGGCACCA TTCTGACCAT GGTGCGTACT GAGGGCCCCC GAAGCCTCTA CAATGGGCTG GTTGCCGGCC TGCAGCGCCA AATGAGCTTT GCCTCTGTCC GCATCGGCCT GTATGATTCT GTCAAACAGT TCTACACCAA GGGCTCTGAG C Intron 4-5 73,688,930 73,688,063   868 gtgagtatgg agcaagggtg taggc...cactg accccatggc tcgcccacag 374 5 ENSE00001 73,688,062 73,687,868 1 1   195 ATGCCAGCAT TGGGAGCCGC CTCCTAGCAG GCAGCACCAC 375 184355 AGGTGCCCTG GCTGTGGCTG TGGCCCAGCC CACGGATGTG GTAAAGGTCC GATTCCAAGC TCAGGCCCGG GCTGGAGGTG GTCGGAGATA CCAAAGCACC GTCAATGCCT ACAAGACCAT TGCCCGAGAG GAAGGGTTCC GGGGCCTCTG GAAAG Intron 5-6 73,687,867 73,687,788    80 gtgtgtacca gttgttttcc cttcc...accca ggatcttcct cctcctacag 376 6 ENSE00003 73,687,787 73,687,686 1 1   102 GGACCTCTCC CAATGTTGCT CGTAATGCCA TTGTCAACTG TGCTGAGCTG 377 147097 GTGACCTATG ACCTCATCAA GGATGCCCTC CTGAAAGCCA ACCTCATGAC AG Intron 6-7 73,687,685 73,686,717   969 gtgagtcatg aggtagacgg tgctg...tgcct tgcctgctcc tccttggcag 378 7 ENSE00001 73,686,716 73,686,536 1 2   181 ATGACCTCCC TTGCCACTTC ACTTCTGCCT TTGGGGCAGG CTTCTGCACC 379 184349 ACTGTCATCG CCTCCCCTGT AGACGTGGTC AAGACGAGAT ACATGAACTC TGCCCTGGGC CAGTACAGTA GCGCTGGCCA CTGTGCCCTT ACCATGCTCC AGAAGGAGGG GCCCCGAGCC TTCTACAAAGG Intron 7-8 73,685,535 73,686,167   369 gtgagcctct ggtcctcccc accca...atgac ctgtgatttt tctcctctag 380 8 ENSE00001 73,685,166 73,685,712 2 —   455 GTTCATGCCC TCCTTTCTCC GCTTGGGTTC CTGGAACGTG GTGATGTTCG 381 184368 TCACCTATGA GCAGCTGAAA CGAGCCCTCA TGGCTGCCTG CACTTCCCGA GAGGCTCCCT TCTGAGCCTC TCCTGCTGCT GACCTGATCA CCTCTGGCTT TGTCTCTAGC CGGGCCATGC TTTCCTTTTC TTCCTTCTTT CTCTTCCCTC CTTCCCTTCT CTCCTTCCCT CTTTCCCCAC CTCTTCCTTC CGCTCCTTTA CCTACCACCT TCCCTCTTTC TACATTCTCA TCTACTCATT GTCTCAGTGC TGGTGGAGTT GACATTTGAC AGTGTGGGAG GCCTCGTACC AGCCAGGATC CCAAGCGTCC CGTCCCTTGG AAAGTTCAGC CAGAATCTTC GTCCTGCCCC CGACAGCCCA GCCTAGCCCA CTTGTCATCC ATAAAGCAAG CTCAACCTTG GCGTC 3′ tcctccctct cttgtagctc ttaccagagg tcttggtcca atggcctttt... 382 downstream sequence

The nucleic acid sequence of the 15,174 base pair (bp) of the human UCP2 gene (ENSG00000175567), including 5,000 bp 5′UTR and 2,000 bp 3′UTR, is as follows (Exons are highlighted) [SEQ ID NO: 383]: 

What is claimed is:
 1. A method for treating diabetes in a subject, the method comprising administering to the subject an effective amount of an antagomir of miR-22, miR-22-3p or miR-22-5p.
 2. The method of claim 1, wherein the human subject selected for treatment is obese or has a genetic or epigenetic predisposition to obesity.
 3. The method of claim 1, wherein the antagomir modulates the activity or expression of UCP1 or UCP2.
 4. The method of claim 1, wherein the miRNA agent comprises an antagomir of miR-22-3p.
 5. The method of claim 1, wherein the miRNA agent is linked to a targeting moiety.
 6. The method of claim 5, wherein the targeting moiety is an aptamer.
 7. The method of claim 5, wherein the targeting moiety delivers the miRNA agent to a specific cell type or tissue.
 8. The method of claim 1, wherein the miRNA agent directly binds to the mRNA or promoter region of at least one mitochondrial uncoupler.
 9. The method of claim 1, wherein the miRNA agent directly binds to the 5′UTR or coding sequence of the mRNA of at least one mitochondrial uncoupler.
 10. The method of claim 1, wherein the miRNA agent modulates the activity of an activator or repressor of a mitochondrial uncoupling protein.
 11. The method of claim 1, wherein the mRNA or protein expression of the mitochondrial uncoupling protein is upregulated.
 12. The method of claim 1, wherein the mitochondrial uncoupling activity of the mitochondrial uncoupling protein is upregulated.
 13. The method of claim 1, wherein the subject is a mammal.
 14. The method of claim 13, wherein the mammal is a human.
 15. The method of claim 1, wherein the diabetes comprises type 2 diabetes mellitus.
 16. The method of claim 15, wherein the type 2 diabetes mellitus is early onset type 2 diabetes mellitus.
 17. A method for increasing insulin sensitivity in a subject in need thereof comprising administering the subject an effective amount of an antagomir of miR-22, miR-22-3p or miR-22-5p.
 18. The method of claim 17, wherein the subject is a mammal.
 19. The method of claim 18, wherein the mammal is a human. 